Chemistry – A European Journal

Riboswitch is a non-coding messenger RNA (m-RNA) whose aptamer domain binds cognate ligands and subsequently undergoes conformational changes in the expression platform leading to the regulation of gene expression. Fluoride riboswitch has immense pharmacological potential due to its presence in some human bacterial pathogens. Several experimental studies shed light on the bacterial defense mechanism of Fluoride riboswitch upon binding of F- cognate ligand in the presence of Mg2+. However, the structural and thermodynamic basis of ligand binding with Fluoride riboswitch aptamer is not well known. This fascinates us for investigating the conformational stability of (i) the holo form of T. Petrophila fluoride riboswitch aptamer (RNA+F-+Mg2++K+) with respect to (ii) the apo form of fluoride riboswitch (RNA in the absence of F- +Mg2++K+). Conformational thermodynamics results derived from molecular dynamics simulation reveal that the holo form of the Fluoride riboswitch aptamer is stabilized by ion recognition site, pseudoknot, and stem1. However, Stem2, Loop1, Loop2, and most of the unpaired bases show significant disorder and destabilization. Molecular docking study validates the thermodynamically destabilized and disordered residues from Loop1 and Stem2 of the Fluoride riboswitch aptamer to serve as putative binding sites for non-cognate ligands. The global health system in the current century faces a serious crisis to counteract bacterial infection due to the severe emergence of bacterial resistance to antibiotics. Consequently, a need for a new generation of antibiotics against resistant bacteria is critically acclaimed. Our work hopefully improves the design of new ligands and aptamers which may be helpful in nucleic acid-targeted therapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/600262v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@145b5beorg.highwire.dtl.DTLVardef@19eeb58org.highwire.dtl.DTLVardef@6d0e6corg.highwire.dtl.DTLVardef@1ce0c10_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIConformational stability of (i) the holo form of T. Petrophila fluoride riboswitch aptamer (RNA+F-+Mg2++K+) with respect to (ii) the apo form of fluoride riboswitch (RNA in the absence of F- +Mg2++K+) is studied. C_LIO_LIHolo Fluoride riboswitch aptamer gets energetically and entropically stable at Pseudoknot, Stem1, and Ion recognition sites whereas Stem2, Loop1, Loop2, and most of the unpaired bases show significant disorder and destabilization. C_LIO_LIThe hydrogen bond network for Pseudoknot, Stem1, and Stem2 in apo Fluoride riboswitch is significantly weak. C_LIO_LIMolecular Docking study confirms that the thermodynamically destabilized and disordered residues from Stem2 and Loop1 of the holo Fluoride riboswitch aptamer serve as putative binding sites for non-cognate ligands. C_LI

Biosynthetic gene clusters (BGC) involved in aryl polyene (APE) biosynthesis are supposed to represent the most widespread BGC in the bacterial world.[1-3] Still, only hydrolysis products[4-8] and not the full-length product(s) have been identified, hindering studies on their biosynthesis and natural function. Here, we apply subsequent chromatographic separations to purify the aryl polyene-containing lipids (APELs) from the entomopathogenic bacterium Xenorhabdus doucetiae. Structure elucidation using a combination of isotope labeling, nuclear magnetic resonance techniques, and tandem mass spectrometry reveals an array of APELs featuring an all-trans C26:5 conjugated fatty acyl and a galactosamine-phosphate-glycerol moiety. In combination with extensive genetic studies, this research broadens the bacterial natural product repertoire and paves the way for future functional characterization of this almost universal microbial compound class. Due to their protective function against reactive oxygen species,[5,9] APELs might be important for virulence or symbiosis, mediating organismic interactions in several ecological niches.

A large series of dipeptides containing sulfur groups and antimony SbV were modeled to understand their inhibitor activity against Leishmaniasis. The trypanothione reductase (TR), which acts as a reducing agent in several vital processes, is responsible for maintaining the parasite’s cellular thiol redox balance. The antimonic SbV acid (Sb2O5·nH2O) is being evaluated as a drug with inhibitory activity against Leishmaniasis. In the present work, we investigated the inhibitory effect of antimony oxide on (TR) activity modeled as a substrate by probing two model clusters in gas phase and continuum water medium: A [(Sb2O10H8)]−2 coordinated to cysteine, and B [Sb7O28H21] coordinated to trypanothione, including glucose adduct. We report here density functional theory (DFT and DFT-D3) using (B3LYP/LANL2DZ and (TPSS/def2TZVP) results on the binding energy of cysteine and trypanthione complexed to these clusters as possible sites promoting the inhibition process. Upon viewing the results of the computational studies of cluster models and theoretical thermochemistry data for receptor-substrate interactions, identification of ligand-cluster interactions helps to unravel the mechanism of inhibition. The acidity of (Sb2O5,nH2O) leads to great cluster-dipeptide passivation. The electrostatic forces between cluster interface and dipeptide interaction present relevant inhibition effects through proton transfer or mobility from the different amine and ketone groups. The reactivity differences come from the unoccupied lone pairs 5pSb which lie at higher energy but remain available to make a good interaction with the lowest orbital p nitrogen in NH2, and in the CO groups substrate fragment in the zone HOMO-LUMO. Further cluster stability comparisons show a lower Gibbs free energy in B3 (B/trypanthione/glucose) (18 – 30 kcal/mol) at both used level in this study and gives good accurate intramolecular interactions, confirmed by the use of the dispersion-corrected density functional (DFT-D3). Given the dipeptide H-mobility and the (Sb2O5,nH2O) cluster acidity, (donor-acceptor duality), the system is predicted to be potent cluster of the inhibitors by endothermic and spontaneous reaction requiring 3.10 kcal/mol in aqueous medium.View Full Text

Large library docking of tangible molecules has revealed potent ligands across many targets. While make-on-demand libraries now exceed 75 billion enumerated molecules, their synthetic routes are dominated by a few reaction types, reducing diversity and inevitably leaving many interesting bioactive-like chemotypes unexplored. Here, we investigate the large-scale enumeration and targeted docking of isoquinuclidines. These "natural-product-like" molecules are rare in the current libraries and are functionally congested, making them interesting as receptor probes. Using a modular, four-component reaction scheme, we built and docked a virtual library of over 14.6 million isoquinuclidines against both the {micro}- and{kappa} -opioid receptors (MOR and KOR, respectively). Synthesis and experimental testing of 18 prioritized compounds found nine ligands with low {micro}M affinities. Structure-based optimization revealed low- and sub- nM antagonists and inverse agonists targeting both receptors. Cryo-electron microscopy (cryoEM) structures illuminate the origins of activity on each target. In mouse behavioral studies, a potent member of the series with joint MOR-antagonist and KOR-inverse-agonist activity reversed morphine-induced analgesia, phenocopying the MOR-selective anti-overdose agent naloxone. Encouragingly, the new molecule induced less severe opioid-induced withdrawal symptoms compared to naloxone during withdrawal precipitation, and did not induce conditioned-place aversion, likely reflecting a reduction of dysphoria due to the compounds KOR-inverse agonism. The strengths and weaknesses of bespoke library docking, and of docking for opioid receptor polypharmacology, will be considered.

DNA-interactions with multivalent ligand(s) have increasingly become the subject of substantial research. For several small-molecules with therapeutic-potential, nucleic-acids serve as their primary molecular-target. Such interaction has been shown to affect transcription, and replication, ultimately leading to apoptotic cell-death. Thus, researchers are becoming increasingly interested in understanding ligand-interaction with oligonucleotides making it possible to develop new, DNA-specific drugs. Yohimbe (Yh), a bioactive indole-alkaloid, has been thoroughly investigated for its pharmaceutical qualities, but the mechanism of DNA-binding is still ambiguous. This research adopted computational and multi-spectroscopic methods to investigate the molecular-mechanism between Yohimbine and hairpin-duplex oligonucleotide at physiological-conditions. The occurrence of slight hypochromic and bathochromic deviations in fluorescence intensity indicates that Yh interacts with hairpin-duplex. Employing the McGhee-von Hipple approach, the Scatchard-plot analyses indicated non-cooperative interaction with 105 M-1 binding affinities. The temperature-dependent fluorescence data suggested positive entropy and negative enthalpy supporting the exothermic binding. Salt-dependent fluorescence revealed that non-polyelectrolytic forces governed the DNA-ligand association. The findings of the urea-denaturation, dye-displacement, and molecular-docking analysis, iodide-quenching confirmed groove-binding. Therefore, biophysical tools and in silico modeling were utilized to identify the structural-alteration and energetic-profiling of Yhs interaction with oligonucleotides which can be employed for the development of DNA-targeted therapeutics.

RNA repeat expansions fold into stable structures and cause microsatellite diseases such as Huntingtons disease (HD), myotonic dystrophy type 1 (DM1), and spinocerebellar ataxias (SCAs). The trinucleotide expansion of r(CAG), or r(CAG)exp, causes both HD and SCA3, and the RNAs toxicity has been traced to its translation into polyglutamine (polyQ; HD) as well as aberrant pre-mRNA alternative splicing (SCA3 and HD). Previously, a small molecule, 1, was discovered that binds to r(CAG)exp and rescues aberrant pre-mRNA splicing in patient-derived fibroblasts by freeing proteins bound to the repeats. Here, we report the structures of single r(CAG) repeat motif (5CAG/3GAC where the underlined adenosines form a 1x1 nucleotide internal loop) in complex with 1 and two other small molecules via nuclear magnetic resonance (NMR) spectroscopy combined with simulated annealing. Compound 2 was designed based on the structure of 1 bound to the RNA while 3 was selected as a diverse chemical scaffold. The three complexes, although adopting different 3D binding pockets upon ligand binding, are stabilized by a combination of stacking interactions with the internal loops closing GC base pairs, hydrogen bonds, and van der Waals interactions. Molecular dynamics (MD) simulations performed with NMR-derived restraints show that the RNA is stretched and bent upon ligand binding with significant changes in propeller-twist and opening. Compound 3 has a distinct mode of binding by insertion into the helix, displacing one of the loop nucleotides into the major groove and affording a rod-like shape binding pocket. In contrast, 1 and 2 are groove binders. A series of single molecule magnetic force spectroscopy studies provide a mechanistic explanation for how bioactive compounds might rescue disease-associated cellular phenotypes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/608150v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@145398org.highwire.dtl.DTLVardef@7c37caorg.highwire.dtl.DTLVardef@132cbbeorg.highwire.dtl.DTLVardef@1de59d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

High-risk neuroblastoma is one of the most lethal childhood cancers. Half of these tumors are driven by MYCN gene amplification (MNA). Despite intensive chemo- and radiotherapy, only 40% of patients survive, and they often suffer from severe long-term side effects of these genotoxic treatments. Thus, new therapies are needed that are less toxic and more efficacious. Here, we identified diphenyleneiodonium (DPI) as a tool compound that preferentially targets MNA neuroblastoma. Using proteomic assays we investigated the DPI mode of action, finding that DPI induces the proteasomal degradation of MYCN and could reverse some alterations induced by high levels of MYCN. These include profound changes in the expression of proteins participating in the mitochondrial electron transport chain. Metabolic and biological assays suggested that alterations in mitochondrial function and the associated production of reactive oxygen species (ROS) are critical DPI targets in the context of MNA. DPI reduced the survival, and malignant transformation of neuroblastoma across a panel of cell lines at clinically achievable concentrations. DPI also shrank tumors and prevented metastatic spread in zebrafish models of MYCN-driven neuroblastoma. These findings suggest that processes impacted by complex I inhibitors could be valuable new targets for the development of non-genotoxic drugs against high-risk MNA neuroblastoma. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/619268v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@167e953org.highwire.dtl.DTLVardef@10749e4org.highwire.dtl.DTLVardef@1842598org.highwire.dtl.DTLVardef@c05d82_HPS_FORMAT_FIGEXP M_FIG C_FIG

Reprogrammed metabolism of cancer cells offers a unique target for pharmacological intervention. In the current study, a series of novel and potentially metabolically stable fluoro-substituted aminocarboxycoumarin derivatives are evaluated for their mitochondrial pyruvate carrier (MPC) inhibition properties. Our studies indicate that the aminocarboxycoumarin template elicits potent MPC inhibitory characteristics, and specifically, structure activity relationship studies show that the N-methyl-N-benzyl structural template provides the optimal inhibitory capacity. Further respiratory experiments demonstrate that candidate compounds specifically inhibit pyruvate driven respiration without substantially affecting other metabolic fuels consistent with MPC inhibition. Further, computational homology and inhibitor docking studies illustrate that aminocarboxycoumarin binding characteristics are indicative of reversible covalent bonding with amino acids in the pyruvate binding domain. Epifluorescent microscopy experiments illustrated that FACC2 accumulates in the mitochondria to a similar extent as parent 7ACC2. Additionally, lead candidate aminocarboxycoumarin derivative D7 elicits cancer cell proliferation inhibition specifically in monocarboxylate transporter 1 (MCT1) expressing 4T1, consistent with its ability to accumulate intracellular lactate. In vivo tumor growth studies illustrate that D7 significantly reduces the tumor burden in two isogeneic murine cell lines 4T1 and 67nr. These studies provide novel MPC inhibitors with potential for anticancer applications.

REV-ERB is a nuclear hormone receptor that plays important role in the regulation of many physiological processes such as circadian clock regulation, inflammation, and metabolism. Despite its importance, few chemical tools are available to study this receptor. In addition, there is no available X-ray crystal structures of REV-ERB bound with synthetic ligands, hampering the development of targeted therapeutics. SR8278 is the only identified synthetic antagonist of REV-ERB. We have performed Gaussian accelerated molecular dynamics (GaMD) simulations to sample the binding pathway of SR8278 and associated conformational changes to REV-ERB. The simulations revealed a novel and more energetically favorable conformational state than the starting conformation. The new conformation allows ligand binding to the orthosteric binding site in a specific orientation. This state is reached after a tryptophan (Trp436) rotameric switch coupled with H3-H6 distance change. We used the newly identified GaMD conformational state in structure-based virtual screening of one million compounds library which led to the identification of novel REV-ERB antagonist. This study is the first that demonstrates a synthetic ligand binding pathway to REV-ERB, which provided important insights into the REV-ERB functional mechanism and lead to the discovery of novel REV-ERB antagonists. This study further emphasizes the power of computational chemistry methods in advancing drug discovery research.

Currently, G-quadruplex structure targeting strategies are considered as a promising anticancer approach. In the search of selective and potent G-quadruplex binders, Here we discuss an analysis of a few chroman derivatives ligands: (A) chroman 7-[2-pyrrolo]-pyrrole-[1,2-a]12H pyrrolino[2,3-b]chroman-4-one, and (C) 4-methyl-7-[2-pyrrolo]-pyrrole[1,2-a]12H pyrrolino[2,3-b]chroman-4-one and their respective borondifluoride complexes B and D as a quadruplex targeting compounds which found to stabilize G-quadruplex structure. To investigate the binding characteristics of these molecules with G-quadruplex vs. duplex selectivity, In vitro biophysical studies were performed by steady-state fluorescence, UV-visible titration, fluorescent TO displacement assay, CD thermal melting, circular dichroism spectroscopy, and cellular imaging by employing both telomeric and PRCC G-quadruplex forming sequences. Our investigation shows that these chromam ligands and their complexes are able to selectively bind and stabilize parallel and mixed hybrid topology of G-quadruplex both In vitro and in cellular conditions. A molecular docking study also suggests the binding of these compounds with G-quadruplex conformation. Collectively our study suggests these chroman complexes as a potentially useful fluorescent chemical product for G-quadruplex specific ligands and expands an option for G-quadruplex targeting ligands.

Widespread resistance to all clinically used antibiotics has sparked investigations into alternative sources for novel and effective antimicrobial agents. Metal-based compounds (metalloantibiotics) have emerged as a promising class of potential antibiotics exhibiting high hit rates against critical bacterial pathogens while not displaying higher toxicity than organic compounds. Here, we describe the exploration of a novel class of non-toxic, Gram-positive acting platinum-based antibacterial agents with micro to nanomolar activity against a range of methicillin and vancomycin-resistant Staphylococcus aureus strains. Structure-activity relationship (SAR) studies revealed that modifications of the core scaffold result in reduced antibacterial activity. Mode of action studies investigations showed that lead compound Pt1 did not impair cell division, RNA, protein, or cell wall synthesis, nor did it affect membrane integrity or potential. Instead, akin to the structurally similar anticancer drug cisplatin (CisPt), Pt1 treatment resulted in reduced DNA staining, visible nucleoid compaction, and activation of DNA damage repair responses. Importantly, we could show that Pt1 is able to interact with and damage DNA directly, resulting in DNA strand breaks and fragmentation. Pt1 activity can be reduced significantly by high amounts of a hydroxyl radical scavenger. Derivative Pt8, which retained DNA-damaging activity but was less potent in terms of antibacterial activity, was not affected by the presence of radical scavengers, suggesting that Pt1 possesses a multimodal mechanism. In line with this observation, no resistance development to Pt1 was observed over the course of 36 passages. Finally, we could demonstrate the in vivo activity of Pt1, which significantly reduced the bacterial load in a murine S. aureus skin infection model. Altogether, these findings shed light on the SAR and antibacterial mode of action of a novel class of platinum metalloantibiotics, validate its in vivo efficacy, and pave the way for further exploration of platinum compounds as novel drug candidates with a highly attractive activity profile.

Vascular Endothelial Growth Factor-A (VEGF-A), a pluripotent cytokine and angiogenic growth factor mediates the switch to an angiogenic phenotype in cancer cells. The interaction of VEGF-A protein with the VEGF receptors (VEGFR-1and VEGFR-2) starts downstream effect that promotes angiogenesis by mediating migration and increasing the permeability of endothelial cells. A cis-regulatory elements consisting of a polypurine/polypyrimidine (pPu/pPy) tract in the proximal 36-bp region (-85 to -50), can participate in the formation of a stable higher order G-quadruplex structure (G4) which is essential for VEGF promoter activity. During cancer progression the VEGF-A G4 succumbs to cellular pressure and fails to maintain the stable structure. This shifts the balance to form duplex structure thereby increasing the rate of transcription. Earlier research has tried to develop small-molecule ligands to target and stabilize G4, however they either lack specificity or non-toxicity. Peptide on the other hand are very less studied. Here we used bioinformatics in-silico tool to develop peptides which can successfully bind and stabilize the VEGF-A G4 while reducing its gene expression. This further alters the expression fate of the VEGF-A signalling cascade and prevents angiogenesis in cancer cells. We used high resolution Nuclear magnetic resonance and molecular dynamics simulation to map the chemistry of the interaction while the qPCR and western blot allowed us to check the expression pattern of the molecules of VEGF-A signalling cascade. In this investigation, we navigate the complex interplay between peptides and quadruplex structures, unravelling valuable insights that can enhance the crafting of pharmacophores directed at the dynamic quadruplex structure. The outcomes of our study are promising, paving the way for progress in the realms of research, characterization, and optimization of peptides binding to G-quadruplexes, with potential implications for therapeutic applications.

Acrylamides are the most commonly used warheads of targeted covalent inhibitors (TCIs) directed at cysteines; however, the reaction mechanisms of acrylamides in proteins remain controversial, particularly for those involving protonated or unreactive cysteines. Using the combined semiempirical quantum mechanics (QM)/molecular mechanics (MM) free energy simulations, we investigated the reaction between afatinib, the first TCI drug for cancer treatment, and Cys797 in the EGFR kinase. Afatinib contains a {beta}-dimethylaminomethyl ({beta}-DMAM) substitution which has been shown to enhance the intrinsic reactivity and potency against EGFR for related inhibitors. Two hypothesized reaction mechanisms were tested. Our data suggest that Cys797 becomes deprotonated in the presence of afatinib and the reaction proceeds via a classical Michael addition mechanism, with Asp800 stabilizing the ion-pair reactant state {beta}-DMAM+/C797- and the transition state of the nucleophilic attack. Our work elucidates an important structure-activity relationship of acrylamides in proteins.

Although the dynamics of telomeres during the life expectancy of normal cells have been extensively studied, there are still some unresolved issues regarding this research field. For example, the conditions required for telomere shortening leading to malignant transformations are not fully understood. In this work, we mass analyzed DNA of normal and cancer cells for comparing telomere isotopic compositions of white blood cells and cancer cells. We have found that the 1327 Da and 1672 Da characteristic telomere mass to charges cause differential mass distributions of about 1 Da for determining isotopic variations among normal cells relative to cancer cells. These isotopic differences are consistent with a prior theory that replacing primordial, common isotopes of 1H, 12C, 14N, 16O, 24Mg, 31P and/or 32S by nonprimordial, uncommon isotopes of 2D, 13C, 15N, 17O, 25Mg and/or 33S leads to altered enzymatic dynamics for modulating DNA and telomere codons towards transforming normal cells to cancer cells. The prior theory and current data are consistent also with a recently observed non-uniform methylation in DNA of cancer cells relative to more uniform methylation in DNA of normal cells. We observe further evidence of nonprimordial isotopic accelerations of acetylations, methylations, hydroxylations and aminations of nucleosides with alterations of phosphorylations of nucleotides for possibly explaining the induced mutations of DNA, RNA and proteins leading to cancer and more general alterations of DNA associated with aging. The different mass spectra of normal and cancer DNA may be reasoned by different functionalizations and isotopic enrichments as causing different motionally induced atomic and nucleotide orders by different nuclear magnetic moments (NMMs); many motionally induced oligonucleotides causing nanoscale disorder and chaos; and the many such motionally induced nanoscale chaoses of different genes causing order in macroscopic DNA for organelles organizations.Competing Interest StatementThe authors have declared no competing interest.View Full Text

The synthesized molybdenum complex, [cis-MoO2(BHAN)2] (BHAN= {beta}-hydroxy--naphthaldehyde), exhibits remarkable efficacy in safeguarding DNA against radiation-induced damage. Comparative studies reveal that the complex offers superior protection to radiolysed DNA compared to the ligand (BHAN). Notably, at a concentration of 2 mM, the complex demonstrates the capability to shield 90% of damaged plasmid DNA from a 20 Gy radiation exposure. Additionally, it also affords significant protection against radiation-induced damage to cellular DNA (CTDNA) from gamma rays. These findings underscore the significant potential of cis-MoO2(BHAN)2 as an effective radioprotector for normal tissues in the context of radiotherapy. The results of this study contribute valuable insights into the advancement of radioprotective strategies, presenting a noteworthy breakthrough with implications for future medical advancements.

Statins form a class of drugs often administered in a variety of cardiovascular diseases, for which their antioxidant capacity appears particularly relevant. Although experiments have long provided empirical evidence that statins can suppress various oxidation pathways, theoretical attempts to quantify the antioxidant activity of statins (read, atorvastatin ATV, because this is the only one studied so far) were not published until last year. Molecular and clinical differences of stains trace back to the ring attached to the statins active moiety. This can be, e.g., a pyrrole, as the case of the aforementioned ATV or a quinoline, as the case of pitavastatin (PVT), which represents the focus of the present work. Extensive results reported here for PVT and derivative include the thermodynamic antioxidant descriptors (bond dissociation enthalpy BDE, adiabatic ionization potential IP, proton dissociation enthalpy PDE, proton affinity PA, and electron transfer enthalpy ETE) related to the three antioxidant mechanisms (hydrogen atom transfer HAT, stepwise electron transfer proton transfer SETPT, sequential proton loss electron transfer SPLET). Our particular emphasis is on the PVTs hydroxylated derivatives wherein a hydroxy group replaces a hydrogen atom either on the quinoline core (Q-hydroxylated metabolites) or on the fluorophenyl ring (F-hydroxylated metabolites). Our calculations indicate that both the Q- and F-hydroxylated metabolites possess antioxidant properties superior to the parent PVT molecule. Given the fact that, to the best of our knowledge, no experimental data for the antioxidant potency of PVT and its hydroxylated derivatives exist, this is a theoretical prediction, and we Given the fact that, to the best of our knowledge, no experimental data for the antioxidant potency of PVT and its hydroxylated derivatives exist, this is a theoretical prediction for the validation of which we aim hereby to stimulate companion experimental in vivo and in vitro investigations and inspire pharmacologists in further drug developments.

Nitazenes are a class of novel synthetic opioids with exceptionally high potency. Currently, an experimental structure of {micro}OR-opioid receptor ({micro}OR) in complex with a nitazene is lacking. Here we used a suite of computational tools, including consensus docking, conventional molecular dynamics (MD) and metadynamics simulations, to investigate the {micro}OR binding modes of nitro-containing meto-, eto-, proto-, buto-, and isotonitazenes and nitro-less analogs, metodes-, etodes-, and protodesnitazenes. Docking generated three binding modes, whereby the nitro-substituted or unsubstituted benzimidazole group extends into SP1 (subpocket 1 between transmembrane helix or TM 2 and 3), SP2 (subpocket 2 between TM1, TM2, and TM7) or SP3 (subpocket 3 between TM5 and TM6). Simulations suggest that etonitazene and likely also other nitazenes favor the SP2-binding mode. Comparison to the experimental structures of {micro}OR in complex with BU72, fentanyl, and mitragynine pseudoindoxyl (MP) allows us to propose a putative model for {micro}OR-ligand recognition in which ligand can access hydrophobic SP1 or hydrophilic SP2, mediated by the conformational change of Gln1242.60. Interestingly, in addition to water-mediated hydrogen bonds, the nitro group in nitazenes forms a{pi} -hole interaction with the conserved Tyr751.39. Our computational analysis provides new insights into the mechanism of {micro}OR-opioid recognition, paving the way for investigations of the structure-activity relationships of nitazenes. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/616560v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@f3a933org.highwire.dtl.DTLVardef@e98f41org.highwire.dtl.DTLVardef@1bc0962org.highwire.dtl.DTLVardef@c416b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Intermediates along the fibrillation pathway are generally considered to be the toxic species responsible for the pathologies of amyloid diseases. However, structural studies of these species have been hampered by heterogeneity and poor stability in standard aqueous conditions. Here, we report a novel methodology for producing stable, on-pathway oligomers of the human Type-2 Diabetes-associated islet amyloid polypeptide (hIAPP, or amylin) using the mechanical forces associated with magic angle spinning (MAS). The species were a heterogeneous mixture of globular and short rod-like species with significant {beta}-sheet content and the capability of seeding hIAPP fibrillation. We used MAS NMR to demonstrate that the nature of the species was sensitive to sample conditions including peptide concentration, ionic strength, and buffer. The methodology should be suitable for studies of other aggregating systems.

Flaviviruses are emerging and re-emerging pathogens of major global health concern that can replicate in hosts of different phyla, including humans. Among them, Zika virus (ZIKV) is associated with severe neurological outcomes, including neonatal microcephaly, congenital malformations, and Guillain-Barre syndrome in adults. Usutu virus (USUV), another mosquito-borne flavivirus, primarily infects birds and has been linked to neurological symptoms in humans. Bagaza virus (BAGV) significantly affects the wildlife of some bird species and carries a noteworthy risk of zoonotic transmission. Despite their clinical significance, approved antiviral options for flaviviral infections (including ZIKV, USUV, and BAGV) are not available, and current management focuses primarily on providing symptomatic relief and supportive care. To address this therapeutic gap, we evaluated the antiviral activity of five phosphaphenalene-based gold (I) complexes. These phosphaphenalene-derived compounds exhibit straightforward and versatile chemistry, allowing access to derivatives with improved stability, bioactivity, selectivity, and cytotoxicity, thereby enhancing their biological activities. Two compounds showed potent inhibition of ZIKV, USUV, and BAGV replication at low micromolar concentrations, with ZIKV displaying greater sensitivity. ZIKV titers were reduced by up to three orders of magnitude in a dose-dependent fashion. Mechanistic studies revealed that both compounds inhibited thioredoxin reductase and disrupted autophagy. This study represents the first demonstration of the potential of phosphaphenalene-based gold (I) complexes as promising candidates against flavivirus infections, offering a promising foundation for the development of urgently needed antiviral therapies with potential impact on global health.

Hydroxysteroid 11-beta dehydrogenase 1 (11{beta}-HSD1) plays a critical role in metabolic homeostasis by catalyzing the intracellular conversion of cortisone to cortisol. Dysregulated 11{beta}-HSD1 activity is closely associated with metabolic disorders such as type 2 diabetes mellitus, obesity, and glucocorticoid-related inflammation. While small-molecule inhibitors of 11{beta}-HSD1 have shown promise, they primarily suppress enzymatic activity without modulating protein abundance. Here, we report the development of the 11{beta}-HSD1-targeting PROTAC degraders. A series of bifunctional molecules were synthesized based on CRBN- and VHL-recruiting ligands, with AZD8329-derived warheads linked via polyethylene glycol chains. Cellular assays demonstrated efficient, ubiquitin-proteasome-dependent degradation of 11{beta}-HSD1, with H-3-V identified as the most potent degrader. In vivo, H-3-V treatment improved glucose tolerance and enhanced glucose-stimulated insulin secretion in a high-fat diet-induced T2DM mouse model. Molecular dynamics simulations revealed that the H-3-V ternary complex exhibited superior binding energetics compared to less active analogs. Collectively, this study introduces a novel chemical modality for 11{beta}-HSD1 modulation and lays the groundwork for future therapeutic development targeting metabolic disease via selective protein degradation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/687522v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@60aa3borg.highwire.dtl.DTLVardef@484120org.highwire.dtl.DTLVardef@1abca2eorg.highwire.dtl.DTLVardef@166b1a3_HPS_FORMAT_FIGEXP M_FIG C_FIG