Synthesis, X-ray Analysis, and Biological Evaluation of a New Class of Stereopure Lactam-Based HIV-1 Protease Inhibitors

In an effort to identify a new class of druglike HIV-1 protease inhibitors, four different stereopure beta-hydroxy gamma-lactam-containing inhibitors have been synthesized, biologically evaluated, and cocrystallized. The impact of the tether length of the central spacer (two or three carbons) was also investigated. A compound with a shorter tether and (3R,4S) absolute configuration exhibited high activity with a K-i of 2.1 nM and an EC50 of 0.64 mu M. Further optimization by decoration of the P1' side chain furnished an even more potent HIV-1 protease inhibitor (K-i = 0.8 nM, EC50 = 0.04 mu M). According to X-ray analysis, the new class of inhibitors did not fully succeed in forming two symmetric hydrogen bonds to the catalytic aspartates. The crystal structures of the complexes further explain the difference in potency between the shorter inhibitors (two-carbon spacer) and the longer inhibitors (three-carbon spacer)

Combining endocannabinoids with retigabine for enhanced M-channel effect and improved KV7 subtype selectivity

Retigabine is unique among anticonvulsant drugs by targeting the neuronal M-channel, which is composed of KV7.2/KV7.3 and contributes to the negative neuronal resting membrane potential. Unfortunately, retigabine causes adverse effects, which limits its clinical use. Adverse effects may be reduced by developing M-channel activators with improved KV7 subtype selectivity. The aim of this study was to evaluate the prospect of endocannabinoids as M-channel activators, either in isolation or combined with retigabine. Human KV7 channels were expressed in Xenopus laevis oocytes. The effect of extracellular application of compounds with different properties was studied using two-electrode voltage clamp electrophysiology. Site-directed mutagenesis was used to construct channels with mutated residues to aid in the mechanistic understanding of these effects. We find that arachidonoyl-L-serine (ARA-S), a weak endocannabinoid, potently activates the human M-channel expressed in Xenopus oocytes. Importantly, we show that ARA-S activates the M-channel via a different mechanism and displays a different KV7 subtype selectivity compared with retigabine. We demonstrate that coapplication of ARA-S and retigabine at low concentrations retains the effect on the M-channel while limiting effects on other KV7 subtypes. Our findings suggest that improved KV7 subtype selectivity of M-channel activators can be achieved through strategically combining compounds with different subtype selectivity.Funding agencies: Swedish Society for Medical Research; Swedish Research CouncilSwedish Research Council [2017-02040]</p

An improved procedure for the synthesis of fourteen 4-OH and 3-MeO-4OH metabolites of fentanyl analogues from two intermediates on multi-gram scale

Fentanyl analogues have appeared on the recreational drug market during the last ten years and caused many fatal overdoses around the world due to their high potencies. Their metabolites are of great interest for toxicology, metabolism and identification studies. According to the literature, fentanyl analogues with similar structures have similar metabolism profile. Therefore, a synthetic route that enables synthesis of the corresponding metabolites for several fentanyl analogues would be valuable. Fentanyl analogue metabolites are often polar and tailing on silica gel. Hence, the purification of these substances could be challengeable. In this work, a general synthetic route was developed and described for the multi-gram scale synthesis of 14 potential metabolites of seven fentanyl analogues. The synthetic route is concise and optimized, does not require any use of silica gel purification and is therefore convenient for large-scale synthesis. The overall yields of the metabolites were in the range of 25-57%.Funding Agencies|Strategic Research Area Forensic Sciences; Swedish Governmental Agency for Innovation SystemsVinnova; Eurostars-2 Joint Programme [E! 113377]; European UnionEuropean Commission</p

Restoration of pure copper motor commutator for aviation by laser powder deposition

The deep grooves often appeared in the commutator of aircraft photoelectric radar motors due to the brush's wear, which resulted in decreased of electrical performance and commutation accuracy, affecting combat performance. Here, we present that the blue laser cladding could be used to repair the faulty part of the pure copper motor commutator. The microstructure, microhardness, wear resistance, and electrical conductivity results show that the surface of the coating was smooth and there were no pores, cracks, and other defects in the bonding interface. There was an apparent transition zone between the matrix and the cladding layer, and a good metallurgical bonding with the matrix was formed. Compared with the matrix, the average microhardness and wear resistance of the cladding layer were increased by 2.22 times and 2.37 times, better than the copper substrate, while the electrical conductivity of the repaired layer was still about 97.7% of that of the pure copper substrate. Our study demonstrates the potential of applying the optimum technological parameters of laser cladding repair to provide the theoretical and experimental basis for mass repair

Searches for Heavy Diboson Resonances at s=13 TeV\sqrt{s} = 13~\mathrm{TeV} with the ATLAS Detector

A combined search for high-mass resonances decaying to $WW$, $WZ$, and $ZZ$ final states with jets in boosted topologies is presented. The combination comprises four individual searches in the $\nu\nu qq$, $\ell\nu qq$, $\ell\ell qq$, and $qqqq$ final states, performed in $pp$ collision data corresponding to an integrated luminosity of $3.2 ~\mathrm{fb}^{-1}$ at $\sqrt{s} = 13~\mathrm{TeV}$ collected by the ATLAS detector at the CERN Large Hadron Collider (LHC). Exclusion limits on the heavy resonance production cross section times branching fraction to dibosons are set at the $95\%$ confidence level for three benchmark signal models. The combined search finds upper exclusion limits on the masses of a scalar singlet, a heavy vector-boson triplet, and a Randall-Sundrum graviton at $2650$, $2600$, and $1100~\mathrm{GeV}$, respectively

Synthesis and identification of an important metabolite of AKB-48 with a secondary hydroxyl group on the adamantyl ring

Studies on the metabolism of bioactive substances containing the adamantyl moiety have shown that hydroxylation is likely to occur at a tertiary carbon of adamantane. Herein, we report the synthesis and identification of one major metabolite of AKB-48, a new illicit psychoactive substance with a hydroxyl group at a secondary carbon of the adamantyl ring. (C) 2017 Elsevier Ltd. All rights reserved.Funding Agencies|National Board of Forensic Medicine in Sweden</p

Metabolism of MMB022 and identification of dihydrodiol formation in vitro using synthesized standards

MMB022 (methyl 3-methyl-2-[1-(pent-4-en-1-yl)-1H-indole-3-carboxamido]butanoate) is a new synthetic cannabinoid with an alkene at the pentenyl side chain, a rare functional group for synthetic cannabinoids. Metabolite identification is an important step for the detection of synthetic cannabinoids in humans, since they are generally extensively metabolized. The aims of the study were to tentatively identify in vitro phase I metabolites, to confirm major metabolites using synthesized metabolites, to examine metabolic pathways thoroughly, to study metabolic stability and to suggest metabolites appropriate for urine screening. MMB022 and its synthesized metabolites were incubated with human liver microsomes (HLM) and the supernatants were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry. Sixteen metabolites were identified, which were generated via dehydrogenation, dihydrodiol formation, ester hydrolysis, hydroxylation, and combinations thereof. A major biotransformation of the alkene at the pentenyl side chain was confirmed to be dihydrodiol formation. The major metabolites were ester hydrolysis (M15) and dihydrodiol (M8) metabolites, whereas the metabolite derived from the combination of ester hydrolysis and dihydrodiol (M5) was the fourth most abundant metabolite. The metabolic pathways were investigated using synthesized metabolites and revealed that M5 is an end product of the pathways, indicating that it might become a more abundant metabolite in vivo depending on the rate of metabolism in humans. The major pathway of MMB022 to M5 was determined to be via M8 formation. Intrinsic clearance of MMB022 was determined to be 296 mL/min/kg andt(1/2)was 2.1 min, indicating a low metabolic stability. M15, M8, and potentially M5 are suggested as suitable urinary targets.Funding Agencies|Eurostars-2 Joint programme [E! 113377]; Strategic Research Area in Forensic Sciences [2016:7]; VINNOVAVinnova [2019-03566]</p

A ratiometric fluorogenic nanoprobe for real-time quantitative monitoring of lysosomal pH

Lysosomes are known as key players in cellular signalling and act as terminal degradation stations involved in a multitude of cellular processes. Being a highly influential physiological factor, pH is essential in the regulation of lysosome-mediated physiological and pathological processes. Aberrant pH fluctuations are highly related to lysosomal dysfunction that correlates to lysosomal storage diseases and neurodegenerative disorders. As such, real-time quantitative monitoring of lysosomal pH (pHL) is crucial for gaining insight into lysosomal dysfunction but challenging by the lack of effective lysosome-specific probes with high signal fidelity. Toward this end, we have proposed a lysosomal fluorogenic nanoprobe (TR-MP) for reliable ratiometric measuring of pHL. It is fabricated by rational manipulation of fluorescence resonance energy transfer (FRET) in a tailorable nanoplatform. The nanoprobe consists of biocompatible silica nanoparticles assembled with a pH-sensitive rhodamine derivative (RDM-TEOS) as an acceptor and aggregation-induced emission (AIE) fluorophore (TPE-OMe) as a donor to ensure high energy transfer efficiency. Further equipped with cell-penetrating facilitator and morpholine to enable effective cell-internalization and high lysosome affinity of TR-MP. Results show that TR-MP can quantitatively measure pH in a range of 3.0 - 7.0 and detect pHL fluctuations in live cells under various stimuli, as well as real-time monitor pHL during apoptosis.Funding Agencies|STINT Joint China-Sweden Mobility Project [CH2017-7243]; Swedish Research Council (VR)Swedish Research Council [VR 2019-02409, 2020-05437]; China Scholarship Council (CSC)China Scholarship Council; Carl Tryggers Stiftelse [CTS 19:379]; Swedish Government strategic faculty grant in material science (SFO, MATLIU) in Advanced Functional Materials (AFM) (VR) [5.1-2015-5959]; Centre in Nano Science and technology at LiTH (CeNano); LiU Cancer network at Linkoping University</p

Transthyretin Binding Mode Dichotomy of Fluorescent trans-Stilbene Ligands

The orientations of ligands bound to the transthyretin (TTR) thyroxine (T4) binding site are difficult to predict. Conflicting binding modes of resveratrol have been reported. We previously reported two resveratrol based trans-stilbene fluorescent ligands, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (SB-11) and (E)-4-(2-(naphthalen-2-yl)vinyl)-benzene-1,2-diol (SB-14), that bind native and misfolded protofibrillar TTR. The binding orientations of these two analogous ligands to native tetrameric TTR were predicted to be opposite. Herein we report the crystal structures of these TTR:ligand complexes. Opposite binding modes were verified but were different than predicted. The reverse binding mode (SB14) placing the naphthalene moiety toward the opening of the binding pocket renders the fluorescent ligand pH sensitive due to changes in Lys15 amine protonation. Conversely, the forward binding mode (SB-11) placing the naphthalene inward mediates a stabilizing conformational change, allowing intersubunit H-bonding between Ser117 of different monomers across the dimer interface. Our structures of TTR complexes answer important questions in ligand design and interpretation of trans-stilbene binding modes to the TTR T4 binding site.Funding Agencies|Goran Gustafssons Foundation; Swedish Research Council [2019-04405]; Swedish Brain Foundation [FO2020-0207]; Knut and Alice Wallenberg Foundation; Carl-Trygger Foundation [CTS2020:479]</p

A drug pocket at the lipid bilayer-potassium channel interface

Many pharmaceutical drugs against neurological and cardiovascular disorders exert their therapeutic effects by binding to specific sites on voltage-gated ion channels of neurons or cardiomyocytes. To date, all molecules targeting known ion channel sites bind to protein pockets that are mainly surrounded by water. We describe a lipid-protein drug-binding pocket of a potassium channel. We synthesized and electrophysiologically tested 125 derivatives, analogs, and related compounds to dehydroabietic acid. Functional data in combination with docking and molecular dynamics simulations mapped a binding site for small-molecule compounds at the interface between the lipid bilayer and the transmembrane segments S3 and S4 of the voltage-sensor domain. This fundamentally new binding site for small-molecule compounds paves the way for the design of new types of drugs against diseases caused by altered excitability.Funding Agencies|Swedish Research Council; Swedish Brain Foundation; Swedish Heart-Lung Foundation; Swedish National Infrastructure for Computing</p