Inhibitor‐Induced Dimerization of an Essential Oxidoreductase from African Trypanosomes

Trypanosomal and leishmanial infections claim tens of thousands of lives each year. The metabolism of these unicellular eukaryotic parasites differs from the human host and their enzymes thus constitute promising drug targets. Tryparedoxin (Tpx) from Trypanosoma brucei is the essential oxidoreductase in the parasite's hydroperoxide‐clearance cascade. In vitro and in vivo functional assays show that a small, selective inhibitor efficiently inhibits Tpx. With X‐ray crystallography, SAXS, analytical SEC, SEC‐MALS, MD simulations, ITC, and NMR spectroscopy, we show how covalent binding of this monofunctional inhibitor leads to Tpx dimerization. Intra‐ and intermolecular inhibitor–inhibitor, protein–protein, and inhibitor–protein interactions stabilize the dimer. The behavior of this efficient antitrypanosomal molecule thus constitutes an exquisite example of chemically induced dimerization with a small, monovalent ligand that can be exploited for future drug design.     

A Suite of “Minimalist” Photo‐Crosslinkers for Live‐Cell Imaging and Chemical Proteomics: Case Study with BRD4 Inhibitors

Affinity‐based probes (Af BPs) provide a powerful tool for large‐scale chemoproteomic studies of drug–target interactions. The development of high‐quality probes capable of recapitulating genuine drug–target engagement, however, could be challenging. “Minimalist” photo‐crosslinkers, which contain an alkyl diazirine group and a chemically tractable tag, could alleviate such challenges, but few are currently available. Herein, we have developed new alkyl diazirine‐containing photo‐crosslinkers with different bioorthogonal tags. They were subsequently used to create a suite of Af BPs based on GW841819X (a small molecule inhibitor of BRD4). Through in vitro and in situ studies under conditions that emulated native drug–target interactions, we have obtained better insights into how a tag might affect the probe's performance. Finally, SILAC‐based chemoproteomic studies have led to the discovery of a novel off‐target, APEX1. Further studies showed GW841819X binds to APEX1 and caused up‐regulation of endogenous DNMT1 expression under normoxia conditions.     

Mirror‐Image Packing Provides a Molecular Basis for the Nanomolar Equipotency of Enantiomers of an Experimental Herbicide

Programs of drug discovery generally exploit one enantiomer of a chiral compound for lead development following the principle that enantiomer recognition is central to biological specificity. However, chiral promiscuity has been identified for a number of enzyme families, which have shown that mirror‐image packing can enable opposite enantiomers to be accommodated in an enzyme's active site. Reported here is a series of crystallographic studies of complexes between an enzyme and a potent experimental herbicide whose chiral center forms an essential part of the inhibitor pharmacophore. Initial studies with a racemate at 1.85 Å resolution failed to identify the chirality of the bound inhibitor, however, by extending the resolution to 1.1 Å and by analyzing high‐resolution complexes with the enantiopure compounds, we determined that both enantiomers make equivalent pseudosymmetric interactions in the active site, thus mimicking an achiral reaction intermediate.     

Synergistic inhibition of SARS-CoV-2 cell entry by otamixaban and covalent protease inhibitors: pre-clinical assessment of pharmacological and molecular properties

SARS-CoV-2, the cause of the COVID-19 pandemic, exploits host cell proteins for viral entry into human lung cells. One of them, the protease TMPRSS2, is required to activate the viral spike protein (S). Even though two inhibitors, camostat and nafamostat, are known to inhibit TMPRSS2 and block cell entry of SARS-CoV-2, finding further potent therapeutic options is still an important task. In this study, we report that a late-stage drug candidate, otamixaban, inhibits SARS-CoV-2 cell entry. We show that otamixaban suppresses TMPRSS2 activity and SARS-CoV-2 infection of a human lung cell line, although with lower potency than camostat or nafamostat. In contrast, otamixaban inhibits SARS-CoV-2 infection of precision cut lung slices with the same potency as camostat. Furthermore, we report that otamixaban''s potency can be significantly enhanced by (sub-) nanomolar nafamostat or camostat supplementation. Dominant molecular TMPRSS2-otamixaban interactions are assessed by extensive 109 μs of atomistic molecular dynamics simulations. Our findings suggest that combinations of otamixaban with supplemental camostat or nafamostat are a promising option for the treatment of COVID-19.

SARS-CoV-2, the cause of the COVID-19 pandemic, exploits host proteins for viral entry into human lung cells and is blocked by otamixaban in combination with a covalent protease inhibitor.     

Universal Approach Towards r‐Hirudin Derivatives with High Anti‐Thrombin Activity Based on Chemical Differentiation of Primary Amino Groups

Chemical modification of recombinant hirudin (r‐hirudin) is necessary whenever surface‐confinement to a biomaterial or biotinylation for subsequent conjugation with carriers is intended. Here, we report a modification strategy that permits chemical discrimination between r‐hirudin's amino groups and preserves its thrombin inhibitor activity. By reaction with Msc‐ONSu, protective groups were successively introduced in r‐hirudin yielding four derivatives (Msc)_x‐hirudin (1 ≤ x ≤ 4) and pure fractions were isolated by ion exchange chromatography. Structure–function relationships were studied for all derivatives and revealed a decrease in activity of more than 90% as compared to unprotected r‐hirudin. MALDI‐TOF MS was used to determine the locations of the Msc groups. Furthermore, evidence was provided that r‐hirudin's N‐terminal amino group is highly important for its anti‐thrombin activity. Selective modification of the lysine residues which maintained the free N‐terminal amino group preserved the anti‐thrombin activity of r‐hirudin even after biotinylation and subsequent linkage to streptavidin or confinement to a polymer surface.     

High-Throughput Label-Free Enzymatic Assays Using Desorption Electrospray-Ionization Mass Spectrometry

High-throughput (HT) enzymatic assays, which typically rely on labeled compounds and plate readers, are important for drug discovery. Mass spectrometry (MS) provides an alternative method of performing HT label-free assays. Here we demonstrate the use of a HT platform based on desorption electrospray ionization (DESI) MS for the label-free study of enzymatic reactions directly from the bioassay matrix with an effective analysis time of 0.3 s per sample. This system allows for thorough analysis of the enzymatic process through monitoring of its substrate and product after an external calibration. We show the platform capabilities by an in-depth study of the acetylcholinesterase assay, including kinetic parameter determination, rapid inhibitor screening, and further characterization of positive hits (that is, IC₅₀ and K_i), as well as inhibition–reactivation assays. We anticipate that the expansion of this platform has high potential impact in label-free enzymology as well as in drug discovery.     

A SAR-based mechanistic study on the combined toxicities of sulfonamides and quorum sensing inhibitors on Escherichia coli

Quorum sensing inhibitors (QSIs) are promising alternatives to antibiotics, but they are discharged into the environment after their use cycle. This poses joint effects on the organisms in the environment. Therefore, it is of great importance to study the combined toxicities of QSIs and antibiotics. In this study, we investigated the single and combined toxicities of four potential QSIs and 11 sulfonamides (SAs) on Escherichia coli. The results revealed that the single toxicities of SAs were greater than those of QSIs, and the toxicities were found positively related to the binding energies (E_bind) with their target proteins, for both antibiotics and QSIs. The combined toxicities of the binary mixtures were observed to be either antagonism or addition. The antagonism could be explained by the phenomenon that QSIs changed SAs molecules into ionic forms, preventing the SA molecules entering the bacteria. Furthermore, it was found that the ratios of the effective concentration (the actual concentration involved in the interaction with the proteins) in the antagonistic cases were higher than those in the additive cases. This study would benefit both rational use of the drug combination and ecological risk assessment of antibiotics and QSIs in the real environment.     

Controlled release of 4-hydroperoxycyclophosphamide from the fatty acid dimer-sebacic acid copolymer

Controlled polymeric release of chemotherapeutic agents has shown promise in the management of malignant gliomas. 4-Hydroperoxycyclophosphamide (4HC), loaded on the fatty acid dimer–sebacic acid copolymer (FAD:SA, 1:1), significantly prolonged survival in rats implanted with F98 and 9L gliomas. Here, we studied the in vitro and in vivo release kinetics in phosphate-buffered saline and rat brain of 20% 4HC/FAD:SA (wt:wt), the optimal dose for treatment of rat gliomas. In vitro release under infinite sink conditions was steady over the initial 12 hr to a peak of 20–35% of impregnated drug, consistent with early phase control via surface erosion. Release over the next 3 weeks was minimal, consistent with barrier formation around the polymer by an oily fatty acid dimer degradation product and consequent slowing of release. However, the polymer started to disintegrate by day 4, and there were minimal visible remnants by 3 weeks. Thus, a considerable amount of polymer-carried drug was probably lost in the disintegrating fragments. Also, drug loss is expected from its inherent hydrolytic instability. In vivo release into brain revealed two peak levels of drug at 0–1 hr and 5–20 days. With loaded polymer implanted intraperitoneally or cyclophosphamide injected systemically, peak brain drug levels were measured in 2–8 hr, with substantial decrease by 48 hr without a second peak. Brain levels were substantially higher than blood levels at all time periods. We conclude that FAD:SA (1:1) adequately protects the otherwise labile 4HC, allowing effective and substained drug release in vivo. Furthermore, it should be possible to modify the polymer to adjust the time of peak release for more beneficial therapeutic effects.     

Inhibition of acrylic acid polymerization by phenothiazine and p-methoxyphenol

The major features of polymerization induction periods for acrylic acid inhibited with phenothiazine and p-methoxyphenol have been characterized at 100°C, including duration of the induction periods, and rates of inhibitor disappearance, molecular oxygen absorption, and peroxide formation. Surprisingly, thermally produced radicals react more rapidly with phenothiazine than with oxygen since there is no detectable oxygen absorption or peroxide for mation during phenothiazine-induced induction periods. Thus, phenothiazine has been used to estimate the thermal rate of radical formation. Phenothiazine's effectiveness as an inhibitor is not directly affected by oxygen, although it does undergo oxygen-promoted, noninhibition-related side reactions. p-Methoxyphenol, on the other hand, depends entirely on the presence of oxygen to function as an inhibitor. Compared with equivalent concentrations of p-methoxyphenol, induction periods obtained with phenothiazine are very long, and the rate of inhibitor disappearance is slower by at least an order of magnitude. The characteristics of p-methoxyphenol inhibition reflect a greater radical flux deriving from the significant rates of oligomeric peroxide formation and decomposition which we measured during p-methoxyphenol-induced induction periods at 100°C. MEHQ is an effective inhibitor at ambient temperatures in part because of the greater stability of the peroxides at these lower temperatures.     

Interactions of a Low Molecular Weight Inhibitor from Streptomyces sp. MBR04 with Human Cathepsin D: Implications in Mechanism of Inactivation

Cathepsin D, a lysosomal aspartic protease, is of potential interest as a target for drug design due to its implication in breast and ovarian cancer. The article reports a low molecular weight cathepsin D inhibitor from Streptomyces sp. MBR04. The M_r of the inhibitor was 1,078 Da as determined by MALDI-TOF, and the amino acid analysis showed the presence of Asp, Asp, Gly, Ala, Lys, Leu, Tyr, Trp residues. The steady-state kinetic interactions revealed reversible, competitive, slow-tight-binding nature of the inhibitor with an IC₅₀ and K _i values of 3.2 and 2.5 nM, respectively. The binding of the inhibitor with the enzyme and the subsequent conformational changes were monitored by exploiting the intrinsic fluorescence of the surface exposed Trp-54 residue. Based on the fluorescence and circular dichroism studies, we demonstrate that the inhibitor binds to the active site of cathepsin D and causes inactivation. All these kinetic, thermodynamic, and quenching studies suggest that the newly isolated peptidic inhibitor could be a potential scaffold to study and can be used to develop new potent therapeutic lead molecule for the development of drugs. The inhibitor will be significant as a potential lead molecule to target cathepsin D.     

Facile synthesis of (±)-, (+)-, and (-)-galanthamine

The Amarylidacea alkaloid galanthamine ( 1a ) is an acetylcholinesterase inhibitor that has been evaluated as a potential agent for the treatment of Alzheimer's disease. We report a very efficent synthesis of (±)-galanthamine [(±)- 1a ] from readily available isovanillin and tyramine. We have separated racemic galanthamine into its diastereoisomeric (1S)-camphanate esters and obtained both natural (-)- and unnatural (+)-galanthamine by lithium aluminum hydride removal of the acyl group.     

Synthesis of the Multidrug Reversal Agent Ko143 and Its Parent Natural Product Fumitremorgin C

Ko143 is a tetracyclic, synthetic analog of the fungal metabolite fumitremorgin C. Ko143 is a potent and specific inhibitor of the membrane-bound efflux transporter ABCG2, and it reverses ABCG2-mediated drug resistance in cancer cells. Here, we describe an improved synthesis of Ko143 that relies on the highly selective, substrate-controlled reduction of an imine that is formed in a Bischler–Napieralski reaction with the amide derived from 6-methoxy-l -tryptophan methyl ester and isovaleric acid as a key step. We have also developed a new route to 6-methoxy-l -tryptophan methyl ester from Cbz-l -aspartic acid methyl ester, m-anisidine and differently substituted benzaldehydes. With p-nitrobenzaldehyde as one of the starting materials, this route gave access to 6-methoxy-l -tryptophan methyl ester in five steps and 20 % overall yield; however, it is less efficient than a previously reported synthesis of 6-methoxy-l -tryptophan methyl ester from 6-methoxy indole.     

Comprehensive Study of the Enhanced Reactivity of Turbo-Grignard Reagents**

Since its introduction in 2004, Knochel's so called Turbo-Grignard reagents revolutionized the usage of Grignard reagents. Through the simple addition of LiCl to a magnesium alkyl an outstanding increase in reactivity can be achieved. Though the exact composition of the reactive species remained mysterious, the reactive mixture itself is readily used not only in synthesis but also found its way into more distant fields like material science. To unravel this mystery, we combined single-crystal X-ray diffraction with in-solution NMR-spectroscopy and closed our investigations with quantum chemical calculations. Using such a variety of methods, we have gained insight into and an explanation for the extraordinary reactivity of this extremely convenient reagent by determining the structure of the first bimetallic reactive species [t-Bu₂Mg ⋅ LiCl ⋅ 4 thf] with two tert-butyl anions at the magnesium center and incorporated lithium chloride.     

Reviews

Abstract

Recent scholarship on Boyle's Sceptical Chymist has emphasised the alchemical context of Boyle's work. In this paper we will draw attention to its specifically sceptical context. Based on Cicero's works on Academic scepticism, the Academica and De Natura Deorum, we give some grounds for Boyle's choice of the literary style of the work and, in particular, for his choice of Carneades as its main character. Based on Sextus Empiricus's Outlines of Pyrrhonism, we show the sceptical nature of the arguments presented by Carneades against the alchemists. Finally, we set Boyle's Sceptical Chymist in the context of Seventeenth-Century skepticism (Gassendi, Mersenne, Descartes, and Glanvill) in order to shed light on the relation exhibited by Boyle's work between scepticism and the new science, in particular the corpuscular theory.     

Structural Insights into Pixatimod (PG545) Inhibition of Heparanase,a Key Enzyme in Cancer and Viral Infections

Pixatimod (PG545), a heparan sulfate (HS) mimetic and anticancer agent currently in clinical trials, is a potent inhibitor of heparanase. Heparanase is an endo-β-glucuronidase that degrades HS in the extracellular matrix and basement membranes and is implicated in numerous pathological processes such as cancer and viral infections, including SARS−CoV-2. To understand how PG545 interacts with heparanase, we firstly carried out a conformational analysis through a combination of NMR experiments and molecular modelling which showed that the reducing end β-D-glucose residue of PG545 adopts a distorted conformation. This was followed by docking and molecular dynamics simulations to study the interactions of PG545 with heparanase, revealing that PG545 is able to block the active site by binding in different conformations, with the cholestanol side-chain making important hydrophobic interactions. While PG545 blocks its natural substrate HS from binding to the active site, small synthetic heparanase substrates are only partially excluded, and thus pentasaccharide or larger substrates are preferred for assaying this class of inhibitor. This study provides new insights for the design of next-generation heparanase inhibitors and substrates.     

Acetylcholinesterase inhibitors: Modeling potential candidates

Alzheimer's disease is the leading cause of dementia for elderly people. The main active therapeutic is supported on the increased levels of acetylcholine in the synaptic cleft, based on reversible inhibition of the acetylcholinesterase (AChE) enzyme. This article aims to propose possible inhibitor candidates for AChE, designed from nonisoprenoid lipids of cashew (Anacardium occidentale), and based on several electronic properties. These electronic properties were obtained through B3LYP/6‐311+G(2d,p) calculation level. Principal component analysis reveals that from the set of studied molecular structures a small group is correlated with donepezil, a drug with known biological activity. © 2012 Wiley Periodicals, Inc.