Quantification of the tissue distribution and accumulation of the neonicotinoid pesticide clothianidin and its metabolites in maternal and fetal mice.

Neonicotinoids (NNs) are commonly used pesticides that have a selective agonistic action on insect nicotinic acetylcholine receptors. Recent evidence has shown that NNs have adverse effects in the next generation of mammals, but it remains unclear how NNs transferred from dams to fetuses are distributed and accumulated in fetal tissues. Here, we aimed to clarify the tissue distribution and accumulation properties of the NN clothianidin (CLO) and its 6 metabolites in 7 tissues and blood in both dams and fetuses of mice administered CLO for a single day or for 9 consecutive days. The results showed that the total concentrations of CLO-related compounds in the brain and kidney were higher in fetuses than in dams, whereas in the liver, heart, and blood they were lower in fetuses. The multi-day administration increased the total levels in heart and blood only in the fetuses of the single administration group. In addition, dimethyl metabolites of CLO showed fetus/dam ratios >1 in some tissues, suggesting that fetuses have higher accumulation property and are thus at higher risks of exposure to CLO-related compounds than dams. These findings revealed differences in the tissue-specific distribution patterns of CLO and its metabolites between dams and fetuses, providing new insights into the assessment of the developmental toxicity of NNs.

Polyhexamethylene guanidine accelerates the macrophage foamy formation mediated pulmonary fibrosis.

Polyhexamethylene guanidine (PHMG) is manufactured and applied extensively due to its superior disinfectant capabilities. However, the inhalatory exposure to PHMG aerosols is increasingly recognized as a potential instigator of pulmonary fibrosis, prompting an urgent call for elucidation of the underlying pathophysiological mechanisms. Within this context, alveolar macrophages play a pivotal role in the primary immune defense in the respiratory tract. Dysregulated lipid metabolism within alveolar macrophages leads to the accumulation of foam cells, a process that is intimately linked with the pathogenesis of pulmonary fibrosis. Therefore, this study examines PHMG's effects on alveolar macrophage foaminess and its underlying mechanisms. We conducted a 3-week inhalation exposure followed by a 3-week recovery period in C57BL/6 J mice using a whole-body exposure system equipped with a disinfection aerosol generator (WESDAG). The presence of lipid-laden alveolar macrophages and downregulation of pulmonary tissue lipid transport proteins ABCA1 and ABCG1 were observed in mice. In cell culture models involving lipid-loaded macrophages, we demonstrated that PHMG promotes foam cell formation by inhibiting lipid efflux in mouse alveolar macrophages. Furthermore, PHMG-induced foam cells were found to promote an increase in the release of TGF-ß1, fibronectin deposition, and collagen remodeling. In vivo interventions were subsequently implemented on mice exposed to PHMG aerosols, aiming to restore macrophage lipid efflux function. Remarkably, this intervention demonstrated the potential to retard the progression of pulmonary fibrosis. In conclusion, this study underscores the pivotal role of macrophage foaming in the pathogenesis of PHMG disinfectants-induced pulmonary fibrosis. Moreover, it provides compelling evidence to suggest that the regulation of macrophage efflux function holds promise for mitigating the progression of pulmonary fibrosis, thereby offering novel insights into the mechanisms underlying inhaled PHMG disinfectants-induced pulmonary fibrosis.

Isolation and characterization of nitroguanidine-degrading microorganisms.

Nitroguanidine (NQ) is a component of newly developed insensitive munition (IM) formulations which are more resistant to impact, friction, heat, or sparks than conventional explosives. NQ is also used to synthesize various organic compounds and herbicides, and has both human and environmental health impacts. Despite the wide application and associated health concerns, limited information is known regarding NQ biodegradation, and only one NQ-degrading pure culture identified as Variovorax strain VC1 has been characterized. Here, we present results for three new NQ-degrading bacterial strains isolated from soil, sediment, and a lab-scale aerobic membrane bioreactor (MBR), respectively. Each of these strains -utilizes NQ as a nitrogen (N) source rather than as a source of carbon or energy. The MBR strain, identified as Pseudomonas extremaustralis strain NQ5, is capable of degrading NQ at a rate of approximately 150 µmole L-1 h-1 under aerobic conditions with glucose as a sole carbon source - and NQ as a sole N source. The addition of NH4+ to strain NQ5 during active growth with NQ as a sole N source slowed the growth rate for several hours, and the strain released NH4+, presumably from NQ. When NO3- was added as an alternate N source under similar conditions, the NO3- was not consumed, but NH4+ release into the culture medium was again observed. Strain NQ5 was also able to utilize guanylurea, guanidine, and ethyl allophanate as N sources, and - tolerate salt concentrations as high as 4 % (as NaCl). The other two stains, NQ4 and NQ7, both identified as Arthrobacter spp., grew significantly slower than strain NQ5 under similar culture conditions and tolerated only ∼1 % NaCl. In addition, neither strain NQ4 nor strain NQ7 was able to degrade guanlyurea or ethyl allophanate, but each degraded guanidine. These strains, particularly strain NQ5, may have practical applications for in-situ and ex-situ NQ bioremediation.

Excited States of apo-Guanidine-III Riboswitch Contribute to Guanidinium Binding through Both Conformational and Induced-Fit Mechanisms.

Riboswitches are mRNA segments that regulate gene expression through conformational changes driven by their cognate ligand binding. The ykkC motif forms a riboswitch class that selectively senses a guanidinium ion (Gdm+) and regulates the downstream expression of proteins which aid in the efflux of excess Gdm+ from the cells. The aptamer domain (AD) of the guanidine-III riboswitch forms an H-type pseudoknot with a triple helical domain that binds a Gdm+. We studied the binding of Gdm+ to the AD of the guanidine (ykkC)-III riboswitch using computer simulations to probe the specificity of the riboswitch to Gdm+ binding. We show that Gdm+ binding is a fast process occurring on the nanosecond time scale, with minimal conformational changes to the AD. Using machine learning and Markov-state models, we identified the excited conformational states of the AD, which have a high Gdm+ binding propensity, making the Gdm+ binding landscape complex exhibiting both conformational selection and induced-fit mechanisms. The proposed apo-AD excited states and their role in the ligand-sensing mechanism are amenable to experimental verification. Further, targeting these excited-state conformations in discovering new antibiotics can be explored.

Mechanisms of NMDA Receptor Inhibition by Sepimostat-Comparison with Nafamostat and Diarylamidine Compounds.

N-methyl-D-aspartate (NMDA) receptors are inhibited by many amidine and guanidine compounds. In this work, we studied the mechanisms of their inhibition by sepimostat-an amidine-containing serine protease inhibitor with neuroprotective properties. Sepimostat inhibited native NMDA receptors in rat hippocampal CA1 pyramidal neurons with IC50 of 3.5 ± 0.3 µM at -80 mV holding voltage. It demonstrated complex voltage dependence with voltage-independent and voltage-dependent components, suggesting the presence of shallow and deep binding sites. At -80 mV holding voltage, the voltage-dependent component dominates, and we observed pronounced tail currents and overshoots evidencing a "foot-in-the-door" open channel block. At depolarized voltages, the voltage-independent inhibition by sepimostat was significantly attenuated by the increase of agonist concentration. However, the voltage-independent inhibition was non-competitive. We further compared the mechanisms of the action of sepimostat with those of structurally-related amidine and guanidine compounds-nafamostat, gabexate, furamidine, pentamidine, diminazene, and DAPI-investigated previously. The action of all these compounds can be described by the two-component mechanism. All compounds demonstrated similar affinity to the shallow site, which is responsible for the voltage-independent inhibition, with binding constants in the range of 3-30 µM. In contrast, affinities to the deep site differed dramatically, with nafamostat, furamidine, and pentamidine being much more active.

Resistance profiles for the investigational neuraminidase inhibitor AV5080 in influenza A and B viruses.

Neuraminidase inhibitors (NAIs) are recommended for influenza treatment and prevention worldwide. The most widely prescribed NAI is oral oseltamivir, while inhaled zanamivir is less commonly used. Using phenotypic neuraminidase (NA) enzymatic assays and molecular modeling approaches, we examined the ability of the investigational orally-dosed NAI AV5080 to inhibit viruses of the influenza A(H1N1)pdm09, A(H3N2), A(H5N1), and A(H7N9) subtypes and the influenza B/Victoria- and B/Yamagata-lineages containing NA substitutions conferring oseltamivir or zanamivir resistance including: NA-R292K, NA-E119G/V, NA-H274Y, NA-I122L/N, and NA-R150K. Broadly, AV5080 showed enhanced in vitro efficacy when compared with oseltamivir and/or zanamivir. Reduced AV5080 inhibition was determined for influenza A viruses with NA-E119G and NA-R292K, and for B/Victoria-lineage viruses with NA-I122N/L and B/Yamagata-lineage virus with NA-R150K. Molecular modeling suggested loss of the short hydrogen bond to the carboxyl group of AV5080 affected inhibition of NA-R292K viruses, whereas loss of the salt bridge with the guanidine group of AV5080 affected inhibition of NA-E119G. The resistance profiles and predicted binding modes of AV5080 and zanamivir are most similar, but dissimilar to those of oseltamivir, in part because of a guanidine moiety compensatory binding effect. Overall, our data suggests that AV5080 is a promising orally-dosed NAI that exhibited similar or superior in vitro efficacy against viruses with reduced or highly reduced inhibition phenotypes with respect to currently approved NAIs.

Mechanism of the extremely high duplex-forming ability of oligonucleotides modified with N-tert-butylguanidine- or N-tert-butyl-N'- methylguanidine-bridged nucleic acids.

Antisense oligonucleotides (ASOs) are becoming a promising class of drugs for treating various diseases. Over the past few decades, many modified nucleic acids have been developed for application to ASOs, aiming to enhance their duplex-forming ability toward cognate mRNA and improve their stability against enzymatic degradations. Modulating the sugar conformation of nucleic acids by substituting an electron-withdrawing group at the 2'-position or incorporating a 2',4'-bridging structure is a common approach for enhancing duplex-forming ability. Here, we report on incorporating an N-tert-butylguanidinium group at the 2',4'-bridging structure, which greatly enhances duplex-forming ability because of its interactions with the minor groove. Our results indicated that hydrophobic substituents fitting the grooves of duplexes also have great potential to increase duplex-forming ability.

Isopropoxy Benzene Guanidine Ameliorates Streptococcus suis Infection In Vivo and In Vitro.

Streptococcus suis, an encapsulated zoonotic pathogen, has been reported to cause a variety of infectious diseases, such as meningitis and streptococcal-toxic-shock-like syndrome. Increasing antimicrobial resistance has triggered the need for new treatments. In the present study, we found that isopropoxy benzene guanidine (IBG) significantly attenuated the effects caused by S. suis infection, in vivo and in vitro, by killing S. suis and reducing S. suis pathogenicity. Further studies showed that IBG disrupted the integrity of S. suis cell membranes and increased the permeability of S. suis cell membranes, leading to an imbalance in proton motive force and the accumulation of intracellular ATP. Meanwhile, IBG antagonized the hemolysis activity of suilysin and decreased the expression of Sly gene. In vivo, IBG improved the viability of S. suis SS3-infected mice by reducing tissue bacterial load. In conclusion, IBG is a promising compound for the treatment of S. suis infections, given its antibacterial and anti-hemolysis activity.

Evaluating neonicotinoid insecticide uptake by plants used as buffers and cover crops.

Runoff and drainage from fields planted with neonicotinoid-coated seeds often contain insecticides that adversely affect aquatic life and other non-target organisms. Management practices such as in-field cover cropping and edge-of-field buffer strips may reduce insecticide mobility, making it important to understand the ability of different plants used in these interventions to absorb neonicotinoids. In this greenhouse study we evaluated uptake of thiamethoxam, a commonly used neonicotinoid, in six plant species - crimson clover, fescue, oxeye sunflower, Maximillian sunflower, common milkweed, and butterfly milkweed - along with a native forb mixture and a native grass plus native forb mixture. All plants were irrigated with water containing 100 or 500 µg/L of thiamethoxam for 60 days, then plant tissues and soils were analyzed for thiamethoxam and its metabolite clothianidin. Crimson clover accumulated up to 50% of the applied thiamethoxam, which was significantly more than other plants and indicates this species may be a hyper-accumulator that can sequester thiamethoxam. In contrast, milkweed plants had relatively low neonicotinoid uptake (<0.5%), meaning that those species may not pose excessive risk to beneficial insects that feed on them. In all plants, accumulated masses of thiamethoxam and clothianidin were greater in above-ground tissues (leaves and stems) than in below-ground roots, with more accrual in leaves than stems. Plants treated with the higher thiamethoxam concentration retained proportionally more of the insecticides. Because thiamethoxam primarily accumulates in above-ground tissues, management strategies that include biomass removal may reduce the input of such insecticides into the environment.

Differential analysis of the dentin soluble proteomic.

OBJECTIVES: To perform a differential analysis of the dentin soluble proteomic and assess the effects of tissue health state and protocol for protein extraction. We hypothesized the dentin soluble proteomic varies according to the tissue physiopathological state (intact vs. caries-affected) and protocol used to extract its proteins. METHODS: Dentin from freshly extracted non-carious and carious teeth were randomly assigned for protein extraction using either guanidine-HCl/ethylenediaminetetraacetic acid (EDTA) or acetic acid. Protein extracts from intact and caries-affected dentin were processed and digested with trypsin for shotgun label-free proteomic analysis (nLC-ESI-MS/MS). Peptides identification was performed on a nanoACQUITY UPLC-Xevo Q-Tof MS system. Peptides identified with scores of confidence greater than 95% were included in the quantitative statistical analysis embedded in the PLGS software. Differences between experimental conditions were calculated using Student test-t with significance pre-set at α=0.05. RESULTS: A total of 158 human proteins were identified. Approximately one-sixth of proteins (24/158) were present in at least two different extracts. Conversely, the greatest number of proteins (134/158) was identified uniquely in only one of the extracts. Overall, a larger number of soluble proteins was retrieved from caries-affected than intact dentin (86/158). Likewise, a greater number of proteins was extracted by the guanidine-HCl/EDTA (106/158) in comparison to acetic acid protocol. Several proteins detected in dentin extracts, mainly those from caries-affected teeth, are biological and/or metabolically involved with tissue turnover/remodeling. CONCLUSION: The identity/abundance of soluble proteins retrieved from and remained in dentin noticeably depend on this tissue physiopathological state and protocol used to remove its minerals. CLINICAL SIGNIFICANCE: The present findings brought new insight into the proteomic phenotype of human dentin and may provide targets for the development of novel caries disease-prevention therapies.

A variant of guanidine-IV riboswitches exhibits evidence of a distinct ligand specificity.

Riboswitches are regulatory RNAs that specifically bind a small molecule or ion. Like metabolite-binding proteins, riboswitches can evolve new ligand specificities, and some examples of this phenomenon have been validated. As part of work based on comparative genomics to discover novel riboswitches, we encountered a candidate riboswitch with striking similarities to the recently identified guanidine-IV riboswitch. This candidate riboswitch, the Gd4v motif, is predicted in four distinct bacterial phyla, thus almost as widespread as the guanidine-IV riboswitch. Bioinformatic and experimental analysis suggest that the Gd4v motif is a riboswitch that binds a ligand other than guanidine. It is found associated with gene classes that differ from genes regulated by confirmed guanidine riboswitches. In inline-probing assays, we showed that free guanidine binds only weakly to one of the tested sequences of the variant. Further tested compounds did not show binding, attenuation of transcription termination, or activation of a genetic reporter construct. We characterized an N-acetyltransferase frequently associated with the Gd4v motif and compared its substrate preference to an N-acetyltransferase that occurs under control of guanidine-IV riboswitches. The substrates of this Gd4v-motif-associated enzyme did not show activity for Gd4v RNA binding or transcription termination. Hence, the ligand of the candidate riboswitch motif remains unidentified. The variant RNA motif is predominantly found in gut metagenome sequences, hinting at a ligand that is highly relevant in this environment. This finding is a first step to determining the identity of this unknown ligand, and understanding how guanidine-IV-riboswitch-like structures can evolve to bind different ligands.

Guanidino acid hydrolysis by the human enzyme annotated as agmatinase.

Guanidino acids such as taurocyamine, guanidinobutyrate, guanidinopropionate, and guanidinoacetate have been detected in humans. However, except for guanidionacetate, which is a precursor of creatine, their metabolism and potential functions remain poorly understood. Agmatine has received considerable attention as a potential neurotransmitter and the human enzyme so far annotated as agmatinase (AGMAT) has been proposed as an important modulator of agmatine levels. However, conclusive evidence for the assigned enzymatic activity is lacking. Here we show that AGMAT hydrolyzed a range of linear guanidino acids but was virtually inactive with agmatine. Structural modelling and direct biochemical assays indicated that two naturally occurring variants differ in their substrate preferences. A negatively charged group in the substrate at the end opposing the guanidine moiety was essential for efficient catalysis, explaining why agmatine was not hydrolyzed. We suggest to rename AGMAT as guanidino acid hydrolase (GDAH). Additionally, we demonstrate that the GDAH substrates taurocyamine, guanidinobutyrate and guanidinopropionate were produced by human glycine amidinotransferase (GATM). The presented findings show for the first time an enzymatic activity for GDAH/AGMAT. Since agmatine has frequently been proposed as an endogenous neurotransmitter, the current findings clarify important aspects of the metabolism of agmatine and guanidino acid derivatives in humans.

Altered Biodistribution and Hepatic Safety Profile of a Gapmer Antisense Oligonucleotide Bearing Guanidine-Bridged Nucleic Acids.

Guanidine-bridged nucleic acid (GuNA) is a novel 2',4'-bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA) analog containing cations that exhibit strong affinity for target RNA and superior nuclease resistance. In this study, Malat1 antisense oligonucleotide (ASO) bearing GuNA was evaluated for target knockdown (KD) activity and tolerability. The GuNA ASO did not interfere with RNase H recruitment on the target RNA/ASO heteroduplex and did show potent target KD activity in a skeletal muscle-derived cell line equivalent to that of the LNA ASO under gymnotic conditions, whereas almost no KD activity was observed in a hepatocyte-derived cell line. The GuNA ASO exhibited potent KD activity in various tissues; the KD activity in the skeletal muscle was equivalent with that of the LNA ASO, but the KD activities in the liver and kidney were clearly lower compared with the LNA ASO. In addition, despite the higher accumulation of the GuNA ASO in the liver, levels of aspartate aminotransferase and alanine aminotransferase with the GuNA ASO administration were not elevated compared with those induced by the LNA ASO. Our data indicate that the GuNA ASO is tolerable and exhibits unique altered pharmacological activities in comparison with the LNA ASO in terms of the relative effect between liver and skeletal muscle.

Discovery of a new flavin N5-adduct in a tyrosine to phenylalanine variant of d-Arginine dehydrogenase.

d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) catalyzes the flavin-dependent oxidation of d-arginine and other d-amino acids. Here, we report the crystal structure at 1.29 Å resolution for PaDADH-Y249F expressed and co-crystallized with d-arginine. The overall structure of PaDADH-Y249F resembled PaDADH-WT, but the electron density for the flavin cofactor was ambiguous, suggesting the presence of modified flavins. Electron density maps and mass spectrometric analysis confirmed the presence of both N5-(4-guanidino-oxobutyl)-FAD and 6-OH-FAD in a single crystal of PaDADH-Y249F and helped with the further refinement of the X-ray crystal structure. The versatility of the reduced flavin is apparent in the PaDADH-Y249F structure and is evidenced by the multiple functions it can perform in the same active site.

Monitoring residue levels and dietary risk assessment of thiamethoxam and its metabolite clothianidin for Chinese consumption of Chinese kale.

BACKGROUND: Thiamethoxam is widely used to control pests in Chinese kale, popularly consumed leafy vegetables. The potential risk to the environment and human health has aroused much public concern. Therefore, it is important to investigate the degradation behavior, residue distribution and dietary risk assessment of thiamethoxam in Chinese kale. RESULTS: A sensitive analytical method for determination of thiamethoxam and its metabolite clothianidin residue in Chinese kale was established and validated through a quick, easy, cheap, effective, rugged, and safe (QuEChERS) technique with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The recoveries were 85.4-101.2% for thiamethoxam and 79.5-108.1% for clothianidin, with the relative standard deviations (RSDs) of 0.9-10.2% and 1.8-6.0%, respectively. For the dissipation kinetics, the data showed that thiamethoxam in Chinese kale was degraded with the half-lives of 4.1 to 4.5 days. In the terminal residue experiments, the residues of thiamethoxam were 0.017-0.357 mg kg-1 after application 2-3 times with a preharvest interval (PHI) of 7 days under the designed dosages. The chronic and acute dietary exposure assessment risk quotient (RQ) values of thiamethoxam in Chinese kale for different Chinese consumers were 0.08-0.19% and 0.05-0.12%, respectively, and those of clothianidin were 0.01-0.04% and 0.02-0.04%, respectively, all of the RQ values were lower than 100%. CONCLUSION: Thiamethoxam in Chinese kale was rapidly degraded following first-order kinetics models. The dietary risk of thiamethoxam and clothianidin through Chinese kale was negligible to consumers. The results from this study are important reference for Chinese governments to developing criteria for the safe and rational use of thiamethoxam, setting maximum residue levels (MRLs), monitoring the quality safety of agricultural products and protecting consumer health. © 2021 Society of Chemical Industry.

In vitro determination of the immunosuppressive effect, internalization, and release mechanism of squalene-gusperimus nanoparticles for managing inflammatory responses.

Gusperimus is an anti-inflammatory drug that has shown to be effective in managing autoimmunity and preventing graft rejection. This is unstable and easily broken down into cytotoxic components. We encapsulated gusperimus binding it covalently to squalene obtaining squalene-gusperimus nanoparticles (Sq-GusNPs). These nanoparticles enhanced the immunosuppressive effect of gusperimus in both mouse macrophages and T cells. The half-maximal inhibitory concentration in macrophages was 9-fold lower for Sq-GusNPs compared with the free drug. The anti-inflammatory effect of the Sq-GusNPs was maintained over time without cytotoxicity. By studying nanoparticles uptake by cells with flow cytometry, we demonstrated that Sq-GusNPs are endocytosed by macrophages after binding to low-density lipoprotein receptors (LDLR). In presence of cathepsin B or D release of gusperimus is increased demonstrating the participation of proteases in the release process. Our approach may allow the application of Sq-GusNPs for effective management of inflammatory disorders including autoimmunity and graft rejection.

Novel [18F]-Labeled Meta-Bromobenzylguanidine Derivatives: Potential Positron Emission Tomography Imaging Probes for the Norepinephrine Transporter.

To develop novel norepinephrine transporter (NET)-targeting positron emission tomography (PET) probes with optimal pharmacokinetic properties, a series of meta-bromobenzylguanidine derivatives was synthesized. 4-Fluorodiethoxyethane-3-bromobenzylguanidine (compound 12) showed relatively good affinity for the NET (IC50 = 1.00 ± 0.04 µM). The corresponding radiotracer 18F-12 was prepared in high radiochemical purity (>98%) via a three-step method. The in vitro cellular uptake results demonstrated that 18F-12 was specifically taken up by NET-expressing SK-N-SH cells by the uptake-1 mechanism. Biodistribution studies in mice showed that 18F-12 exhibited high cardiac uptake (10.45 ± 0.66 %ID/g at 5 min p.i. and 6.44 ± 0.40 %ID/g at 120 min p.i.), faster liver clearance, and a lower dose of absorbed radiation than [123I]-labeled meta-iodobenzylguanidine ([123I]MIBG). Small animal PET imaging confirmed the high heart-to-background ratio of 18F-12 and the uptake-1 mechanism specific for the NET in rats, indicating its potential as a promising PET radiotracer for cardiac sympathetic nerve imaging.

OFF-State-Specific Inhibition of the Proprotein Convertase Furin.

The pro-protein convertase furin is a highly specific serine protease involved in the proteolytic maturation of many proteins in the secretory pathway. It also activates surface proteins of many viruses including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furin inhibitors effectively suppress viral replication and thus are promising antiviral therapeutics with broad application potential. Polybasic substrate-like ligands typically trigger conformational changes shifting furin's active site cleft from the OFF-state to the ON-state. Here, we solved the X-ray structures of furin in complex with four different arginine mimetic compounds with reduced basicity. These guanylhydrazone-based inhibitor complexes showed for the first time an active site-directed binding mode to furin's OFF-state conformation. The compounds undergo unique interactions within the S1 pocket, largely different compared to substrate-like ligands. A second binding site was identified at the S4/S5 pocket of furin. Crystallography-based titration experiments confirmed the S1 site as the primary binding pocket. We also tested the proprotein convertases PC5/6 and PC7 for inhibition by guanylhydrazones and found an up to 7-fold lower potency for PC7. Interestingly, the observed differences in the Ki values correlated with the sequence conservation of the PCs at the allosteric sodium binding site. Therefore, OFF-state-specific targeting of furin can serve as a valuable strategy for structure-based development of PC-selective small-molecule inhibitors.

An uncommon [K+(Mg2+)2] metal ion triad imparts stability and selectivity to the Guanidine-I riboswitch.

The widespread ykkC-I riboswitch class exemplifies divergent riboswitch evolution. To analyze how natural selection has diversified its versatile RNA fold, we determined the X-ray crystal structure of the Burkholderia sp. TJI49 ykkC-I subtype-1 (Guanidine-I) riboswitch aptamer domain. Differing from the previously reported structures of orthologs from Dickeya dadantii and Sulfobacillus acidophilus, our Burkholderia structure reveals a chelated K+ ion adjacent to two Mg2+ ions in the guanidine-binding pocket. Thermal melting analysis shows that K+ chelation, which induces localized conformational changes in the binding pocket, improves guanidinium-RNA interactions. Analysis of ribosome structures suggests that the [K+(Mg2+)2] ion triad is uncommon. It is, however, reminiscent of metal ion clusters found in the active sites of ribozymes and DNA polymerases. Previous structural characterization of ykkC-I subtype-2 RNAs, which bind the effector ligands ppGpp and PRPP, indicate that in those paralogs, an adenine responsible for K+ chelation in the Burkholderia Guanidine-I riboswitch is replaced by a pyrimidine. This mutation results in a water molecule and Mg2+ ion binding in place of the K+ ion. Thus, our structural analysis demonstrates how ion and solvent chelation tune divergent ligand specificity and affinity among ykkC-I riboswitches.

Identification of 13 Guanidinobenzoyl- or Aminidinobenzoyl-Containing Drugs to Potentially Inhibit TMPRSS2 for COVID-19 Treatment.

Positively charged groups that mimic arginine or lysine in a natural substrate of trypsin are necessary for drugs to inhibit the trypsin-like serine protease TMPRSS2 that is involved in the viral entry and spread of coronaviruses, including SARS-CoV-2. Based on this assumption, we identified a set of 13 approved or clinically investigational drugs with positively charged guanidinobenzoyl and/or aminidinobenzoyl groups, including the experimentally verified TMPRSS2 inhibitors Camostat and Nafamostat. Molecular docking using the C-I-TASSER-predicted TMPRSS2 catalytic domain model suggested that the guanidinobenzoyl or aminidinobenzoyl group in all the drugs could form putative salt bridge interactions with the side-chain carboxyl group of Asp435 located in the S1 pocket of TMPRSS2. Molecular dynamics simulations further revealed the high stability of the putative salt bridge interactions over long-time (100 ns) simulations. The molecular mechanics/generalized Born surface area-binding free energy assessment and per-residue energy decomposition analysis also supported the strong binding interactions between TMPRSS2 and the proposed drugs. These results suggest that the proposed compounds, in addition to Camostat and Nafamostat, could be effective TMPRSS2 inhibitors for COVID-19 treatment by occupying the S1 pocket with the hallmark positively charged groups.