Rootstock vigor dictates the canopy light environment that regulates metabolite profile and internal fruit quality development in peach.

Five rootstock cultivars of differing vigor: vigorous ('Atlas™' and 'Bright's Hybrid® 5'), standard ('Krymsk® 86' and 'Lovell') and dwarfing ('Krymsk® 1') grafted with 'Redhaven' as the scion were studied for their impact on productivity, mid-canopy photosynthetic active radiation transmission (i.e., light availability) and internal fruit quality. Αverage yield (kg per tree) and fruit count increased significantly with increasing vigor (trunk cross sectional area, TCSA). Α detailed peach fruit quality analysis on fruit of equal maturity (based on the index of absorbance difference, IAD) coming from trees with equal crop load (no. of fruit cm-2 of TCSA) characterized the direct impact of rootstock vigor on peach internal quality [dry matter content (DMC) and soluble solids concentration (SSC)]. DMC and SSC increased significantly with decreasing vigor and increasing light availability, potentially due to reduced intra-tree shading and better light distribution within the canopy. Physiologically characterized peach fruit mesocarp was further analyzed by non-targeted metabolite profiling using gas chromatography mass spectrometry (GC-MS). Metabolite distribution was associated with rootstock vigor class, mid-canopy light availability and fruit quality characteristics. Fructose, glucose, sorbose, neochlorogenic and quinic acids, catechin and sorbitol were associated with high light environments and enhanced quality traits, while sucrose, butanoic and malic acids related to low light conditions and inferior fruit quality. These outcomes show that while rootstock genotype and vigor are influencing peach tree productivity and yield, their effect on manipulating the light environment within the canopy also plays a significant role in fruit quality development.

Computational drug repositioning identifies niclosamide and tribromsalan as inhibitors of Mycobacterium tuberculosis and Mycobacterium abscessus.

Tuberculosis (TB) is still a major global health challenge, killing over 1.5 million people each year, and hence, there is a need to identify and develop novel treatments for Mycobacterium tuberculosis (M. tuberculosis). The prevalence of infections caused by nontuberculous mycobacteria (NTM) is also increasing and has overtaken TB cases in the United States and much of the developed world. Mycobacterium abscessus (M. abscessus) is one of the most frequently encountered NTM and is difficult to treat. We describe the use of drug-disease association using a semantic knowledge graph approach combined with machine learning models that has enabled the identification of several molecules for testing anti-mycobacterial activity. We established that niclosamide (M. tuberculosis IC90 2.95 µM; M. abscessus IC90 59.1 µM) and tribromsalan (M. tuberculosis IC90 76.92 µM; M. abscessus IC90 147.4 µM) inhibit M. tuberculosis and M. abscessus in vitro. To investigate the mode of action, we determined the transcriptional response of M. tuberculosis and M. abscessus to both compounds in axenic log phase, demonstrating a broad effect on gene expression that differed from known M. tuberculosis inhibitors. Both compounds elicited transcriptional responses indicative of respiratory pathway stress and the dysregulation of fatty acid metabolism.

Revisiting anthelmintic resistance in sheep flocks from São Paulo State, Brazil.

Haemonchus contortus and Trichostrongylus colubriformis are the most important gastrointestinal nematodes causing serious losses in sheep production of tropical and subtropical regions. Prophylaxis of gastrointestinal nematode infections is based on anthelmintics use, but their frequent administration selects multiple-resistant parasites. To evaluate how the situation has changed over the last decades, the anthelmintic resistance status of gastrointestinal nematodes in sheep flocks was assessed in the current study and compared to previous surveys. In each one of the 15 flocks evaluated, animals (n ≥ 7) were allocated into at least five groups and treated as follows: 1) untreated control; 2) albendazole; 3) levamisole; 4) ivermectin; and 5) monepantel. If more animals were available, two additional groups were included: 6) closantel, and 7) moxidectin. The faecal egg count reduction test (FECRT) was carried out to evaluate the pre- and post-treatment using the SHINY tool. Haemonchus spp. was the most prevalent nematode from faecal cultures. The mean efficacy of albendazole was 40%. Only in two farms, levamisole presented a relatively high percentage of reduction in the FECRT about 90%, while ivermectin and moxidectin presented the worst mean efficacy of 34% and 21% among all farms, respectively. Like other anthelmintics, closantel demonstrated low efficacy (63%) across all farms evaluated. Monepantel presented an overall mean efficacy of 79%, but it was the only anthelmintic that presented efficacy ≥95%, in five farms. The results revealed that gastrointestinal nematodes with multiple anthelmintic resistance were prevalent in all 15 sheep herds. The research suggests that nematodes are becoming more and more resistant to various anthelmintic compounds, which has made the problem worse. This circumstance highlights the necessity to put into practice sustainable and long-lasting methods to prevent gastrointestinal nematode infections in sheep husbandry.

Augmentation of musculoskeletal soft tissue morphology within low back pain patients may suggest the presence of physiological stress shielding: An in vivo study.

INTRODUCTION: The pathomechanism of low back pain (LBP) remains unknown. However, changes to mechanical properties of soft tissues affected by LBP may indicate the presence of stress shielding, which may manifest via tissue remodeling. This study investigates the potential for physiological stress shielding within the lumbar spine by examining differences within lumbar soft tissue morphology between control and LBP subjects. METHODS: Through MRI, the total and functional cross-sectional area (tCSA, fCSA) and fatty infiltration (FI) of the lumbar multifidus (MF), erector spinae (ES), quadratus lumborum (QL), psoas major (PM), and thoracolumbar fascia (TLF) were measured from the L1/L2 to L5/S1 intervertebral disc levels of 69 subjects (36 LBP and 33 control subjects). Statistical analysis was conducted using Mann-Whitney U. P < 0.05 denoted significance. RESULTS: Comparison of male LBP patients and male healthy controls yielded an increase in tCSA and fCSA within the L4/L5 PM (p < 0.01), and the L4/L5 ES (p = 0.02) and PM (p < 0.01), respectively, of LBP patients. Female LBP patients' FI compared to female controls increased within the L1/L2 MF (p = 0.03), L3/L4 MF (p = 0.04) and ES (p = 0.02), and L4/L5 QL (p = 0.01). The L3/L4 TLF also demonstrated an 8% increase in LBP subjects. CONCLUSION: Male patients' results suggest elevated tissue loading during motion yielding hypertrophy in the L4/L5 ES and PM fCSA, and PM tCSA. Female LBP patients' MF, ES, and PM at L3/L4 demonstrating elevated FI coupled with TLF tCSA hypertrophy may suggest irregular stress distributions and lay the foundation for stress shielding within musculoskeletal soft tissues.

Effects of Cervical Paravertebral Extensors in Patients with Cervical Ossification of the Posterior Longitudinal Ligament Grouped According to mK-Line.

OBJECTIVES: The objective of this study was to quantify the morphology, composition, and asymmetry of the paravertebral extensor muscles (PSEMs) in patients with cervical ossification of the posterior longitudinal ligament (OPLL) who had different modified K-line (mK-line) and the minimum interval between the mK-line and OPLL (INTmin ) values and to investigate the relationship between PSEMs and symptoms and outcomes following laminoplasty. These original findings elucidated that the atrophy of PSEMs could predict decompression outcomes and provided a theoretical basis for paraspinal muscle rehabilitation. METHODS: A total of 94 consecutive patients who underwent laminoplasty for OPLL between January 2020 and January 2022 were enrolled in this retrospective study. The relative cross-sectional areas (rCSA), functional cross-sectional areas (rFCSA), and FCSA/CSA ratio of the multifidus (MF), semispinalis cervicalis (SSCe), semispinalis capitis (SSCa), and splenius capitis (SpCa) were measured at the C3-C7 segments on cervical magnetic resonance imaging (MRI). This study compared the differences between the mK-line (+) group and the mK-line (-) group, as well as between the INTmin <4 mm group and the INTmin ≥4 mm group, using the independent t-test or Mann-Whitney test for continuous variables and the χ2 -test for categorical variables. The correlations between the PSEMs and symptoms were analyzed using either the Pearson or Spearman correlation coefficient. RESULTS: The relative total CSA (rTCSA) of the PSEMs, especially the MF, was significantly smaller in the mK-line (-) group. However, the FCSA/CSA of the right deep extensor muscle (DEM) was larger. The asymmetry of the MF TFCSA/TCSA showed a significant difference between the mK-line groups. In the INTmin <4 mm group, the PSEMs rCSA and rFCSA were significantly smaller, while the bilateral MF TFCSA/TCSA and right SSCe TFCSA/TCSA were larger. The asymmetry of the superficial extensor muscle rCSA was significantly lower in the group with INTmin <4 mm. The postoperative modified Japanese Orthopedic Association score (mJOA) and mJOA recovery rate were positively correlated with the INTmin and DEM rCSA and negatively correlated with the asymmetry of MF FCSA/CSA. CONCLUSIONS: In patients with mK-line (-) or INTmin <4 mm, the PSEMs were smaller, and the DEM atrophy and composition changes were predominant. The MF asymmetry was higher in patients with mK-lines (-), whereas the SEM atrophy and asymmetry were more prevalent in patients with INTmin <4 mm. The DEM was related to the preoperative and postoperative mJOA scores. DEM-preserving surgery or DEM-specific rehabilitation exercises can improve the recovery of patients with OPLL during the perioperative period. In addition, attention should be paid to the evaluation of the SEM, especially the SpCa at the C3 and C5 levels.

Optimization and validation of high-performance liquid chromatography using modified QuEChERS to determine anthelmintic drugs in mutton.

The aim of this study was to rapidly determine the presence of anthelmintic drugs in sheep meat using the optimized high-performance liquid chromatography-ultraviolet (HPLC-UV) method with modified QuEChERS (quick, easy, cheap, effective, rugged, safe) technology. Fifty fresh sheep meat samples from different slaughterhouses were collected. A double extraction procedure (QuEChERS/HPLC-UV technology) was used to extract the target analytes. A multilevel calibration curve from 1 to 1000 g/kg was used to establish instrument linearity for rafoxanide, albendazole, and closantel, whereas 0.1-100 µg/kg was used for ivermectin, levamisole, and oxyclozanide to find the lowest concentration, maximum residue limit (MRL), and occupied range for targeted analytes. The concentration levels were used to investigate the linearity, whereas several certified reference materials were applied to determine accuracy. The process was linear for all combinations, from the limit of quantification (LOQ) to the maximum concentration. The LOQ was established at 0.5 µg/kg for ivermectin, levamisole, and oxyclozanide and 10 µg/kg for rafoxanide, albendazole, and closantel. Recovery values were 70%-120%, and repeatability/reproducibility stated in relative standard deviation was obtained at less than 20%. QuEChERS method revealed that most meat samples contained anthelmintic drug residues, of which the majority exceeded the MRLs. Thus, the drugs should be used correctly in animals to avoid residues in food for human consumption.

[Toxic retinopathy caused by veterinary antiparasitic closantel: a case report].

A 32-year-old female patient presented with bilateral vision loss for 2 months following her intake of various antiparasitic drugs, including closantel, a veterinary drug, for a self-perceived intraocular parasitic infection. Swept-source optical coherence tomography revealed diffuse hyperreflectivity between the outer nuclear layer and the retinal pigment epithelium, as well as the largely indistinguishable outer retinal layers. This case was clinically diagnosed with veterinary closantel-induced toxic retinopathy and had a poor visual prognosis after nerve nutrition and circulation improvement therapy due to the long duration of the disease.

Design, synthesis and biological evaluation of salicylanilides as novel allosteric inhibitors of human pancreatic lipase.

Obesity is a growing global health problem and is associated with increased prevalence of many metabolic disorders, including diabetes, hypertension and cardiovascular disease. Pancreatic lipase (PL) has been validated as a key target for developing anti-obesity agents, owing to its crucial role in lipid digestion and absorption. In the past few decades, porcine PL (pPL) is always used as the enzyme source for screening PL inhibitors, which generate numerous pPL inhibitors but the potent inhibitors against human PL (hPL) are rarely reported. Herein, a series of salicylanilide derivatives were designed and synthesized, while their anti-hPL effects were assayed by a fluorescence-based biochemical approach. To investigate the structure-activity relationships of salicylanilide derivatives as hPL inhibitors in detail, structural modifications on three rings (A, B and C) of the salicylanilide skeleton were performed. Among all tested compounds, 2t and 2u were found possessing the most potent anti-PL activity, showing IC50 values of 1.86 µM and 1.63 µM, respectively. Inhibition kinetic analyses suggested that both 2t and 2u could effectively inhibit hPL in a non-competitive manner, with the ki value of 1.67 µM and 1.70 µM, respectively. Fluorescence quenching assays suggested that two inhibitors could quench the fluorescence of hPL via a static quenching procedure. Molecular docking simulations suggested that 2t and 2u could tightly bind on an allosteric site of hPL. Collectively, the structure-activity relationships of salicylanilide derivatives as hPL inhibitors were carefully investigated, while two newly identified reversible hPL inhibitors (2t and 2u) could be used as promising lead compounds to develop novel anti-obesity drugs.

The first fluorescent sensor for the detection of closantel in meat.

Closantel is widely used in the management of parasitic infestation in livestock, but is contraindicated in humans due to its high toxic to human retina. Thus, development of a fast and selective method for the detection of closantel residues in animal products is highly needed yet still challenging. In the present study, we report a supramolecular fluorescent sensor for closantel detection through a two-step screening process. The fluorescent sensor can detect closantel with a fast response (<10 s), high sensitivity, and high selectivity. The limit of detection is 0.29 ppm, which is much lower than the maximum residue level set by government. Moreover, the applicability of this sensor has been demonstrated in commercial drugs tablets, injection fluids, and real edible animal products (muscle, kidney, and liver). This work provides the first fluorescence analytical tool for accurate and selective determination of closantel, and may inspire more sensor design for food analysis.

Closantel toxicity.

Closantel is a broad-spectrum antihelminthic agent. It is a veterinary drug used only in animals-usually cattle, sheep and goats. A man in his 60s accidentally ingested approximately 1500 mg closantel. His visual acuity deteriorated. Optical coherence tomography (OCT) showed disruption of the outer retinal layers. Electroretinography identified abnormalities in macula and inner retinal function. He received methylprednisolone 1 g daily intravenously for 3 days. Improvements in both his visual acuity and OCT appearance followed. This case illustrates the profoundly destructive effect of this drug on humans even when consumed in low dose. We provide a concise summary of the small number of cases of closantel toxicity in humans, previously reported, for future reference as needed by others.

Salicylanilides and Their Anticancer Properties.

Salicylanilides are pharmacologically active compounds with a wide spectrum of biological effects. Halogenated salicylanilides, which have been used for decades in human and veterinary medicine as anthelmintics, have recently emerged as candidates for drug repurposing in oncology. The most prominent example of salicylanilide anthelmintic, that is intensively studied for its potential anticancer properties, is niclosamide. Nevertheless, recent studies have discovered extensive anticancer potential in a number of other salicylanilides. This potential of their anticancer action is mediated most likely by diverse mechanisms of action such as uncoupling of oxidative phosphorylation, inhibition of protein tyrosine kinase epidermal growth factor receptor, modulation of different signaling pathways as Wnt/ß-catenin, mTORC1, STAT3, NF-κB and Notch signaling pathways or induction of B-Raf V600E inhibition. Here we provide a comprehensive overview of the current knowledge about the proposed mechanisms of action of anticancer activity of salicylanilides based on preclinical in vitro and in vivo studies, or structural requirements for such an activity.

Closantel retinal toxicity: Recovery from severe vision loss after corticosteroid therapy.

PURPOSE: To present a relatively rare case of retinal toxicity and consequent severe vision loss due to Closantel ingestion. CASE REPORT: A 37-year-old female presented with sudden painless decrease vision in both eyes. She had no previous history of medical disease and denied any trauma. The patient had accidentally ingested Closantel a few days prior to presentation. Closantel is a veterinary anti-helminthic drug used mainly in livestock. Best corrected visual acuity (BCVA) at presentation was 20/200 bilaterally. There was no relative afferent pupillary defect (RAPD) and red saturation test was normal. Macular optical coherence tomography (OCT) revealed disruption in the outer retinal layer and ellipsoid zone in both eyes. A diagnosis of retinal toxicity due to Closantel was made and the patient was started on 1 mg/kg oral prednisolone acetate. On the 45th day after presentation, her BCVA had improved to 20/20 bilaterally. CONCLUSION: Closantel is a potentially toxic drug causing destruction of the neurosensory retina and visual disturbances. We suggest eye-care personnel awareness regarding the risk of Closantel-induced retinal toxicity and prompt treatment with systemic steroids should be considered.

Design and synthesis of novel halogen rich salicylanilides as potential antileishmanial agents.

The available therapeutic treatment for leishmaniasis is inadequate and toxic due to side effects, expensive and emergence of drug resistance. Affordable and safe antileishmanial agents are urgently needed and toward this objective, we synthesized a series of 32 novel halogen rich salicylanilides including niclosamide and oxyclozanide and investigated their antileishmanial activity against amastigotes of Leishmania donovani. In vitro data showed fifteen compounds inhibited intracellular amastigotes with an IC50 of below 5 µM and selectivity index above 10. Among 15 active compounds, 14 and 24 demonstrated better activity with an IC50 of 2.89 µM and 2.09 µM respectively and selectivity index is 18. Compound 24 exhibited significant in vivo antileishmanial efficacy and reduced 65% of the splenic parasite load on day 28th post-treatment in the experimental visceral leishmaniasis golden hamster model. The data suggest that 24 can be a promising lead candidate possessing potential to be developed into a leishmanial drug candidate.

Determination of closantel enantiomers in black goat plasma and their pharmacokinetic characteristics.

Closantel is a broad-spectrum antiparasitic drug widely used in livestock. It is a chiral drug consisting of a pair of enantiomers. An accurate, selective, and validated method was established for separating and detecting closantel enantiomers in black goat plasma. Separation of enantiomers was achieved in Chiralpak AD-3 based on amylose tris (3,5-dimethyl phenyl carbamates) under a gradient mobile phase composed of n-hexane-TFA (100:0.1, v/v) and IPA-MeOH-TFA (99:1:0.1, v/v/v). Mean recoveries of the two enantiomers at three spiking levels ranged from 97.4 % to 102.0 % with a relative standard deviation ≤ 4.5 %. The limit of quantification was 1.35 µg/mL for each enantiomer in plasma. The proposed method was successfully applied to investigate the enantioselective pharmacokinetics of closantel enantiomers (E1 and E2) in black goats following an intramuscular closantel sodium at 5 mg/kg b.w. The results revealed that peak plasma concentration and area under the curve were significantly different between the two enantiomers (p < 0.05). Cmax and AUC0-∞ for closantel E1 were approximately 3 times greater than closantel E2. These findings would help in further evaluation of pharmacokinetic, pharmacodynamic, and toxicity of the individual enantiomers of closantel in food animals.

Study of Biological Activities and ADMET-Related Properties of Salicylanilide-Based Peptidomimetics.

A series of eleven benzylated intermediates and eleven target compounds derived from salicylanilide were tested against Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 as reference strains and against three clinical isolates of methicillin-resistant S. aureus (MRSA) and three isolates of vancomycin-resistant E. faecalis. In addition, the compounds were evaluated against Mycobacterium tuberculosis H37Ra and M. smegmatis ATCC 700084. The in vitro cytotoxicity of the compounds was assessed using the human monocytic leukemia cell line THP-1. The lipophilicity of the prepared compounds was experimentally determined and correlated with biological activity. The benzylated intermediates were found to be completely biologically inactive. Of the final eleven compounds, according to the number of amide groups in the molecule, eight are diamides, and three are triamides that were inactive. 5-Chloro-2-hydroxy-N-[(2S)- 4-(methylsulfanyl)-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino}butan-2-yl]benzamide (3e) and 5-chloro-2-hydroxy-N-[(2S)-(4-methyl-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino)pentan-2-yl)benzamide (3f) showed the broadest spectrum of activity against all tested species/isolates comparable to the used standards (ampicillin and isoniazid). Six diamides showed high antistaphylococcal activity with MICs ranging from 0.070 to 8.95 µM. Three diamides showed anti-enterococcal activity with MICs ranging from 4.66 to 35.8 µM, and the activities of 3f and 3e against M. tuberculosis and M. smegmatis were MICs of 18.7 and 35.8 µM, respectively. All the active compounds were microbicidal. It was observed that the connecting linker between the chlorsalicylic and 4-CF3-anilide cores must be substituted with a bulky and/or lipophilic chain such as isopropyl, isobutyl, or thiabutyl chain. Anticancer activity on THP-1 cells IC50 ranged from 1.4 to >10 µM and increased with increasing lipophilicity.

De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma.

Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH's ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.

Drug screening approach against mycobacterial fatty acyl-AMP ligase FAAL32 renews the interest of the salicylanilide pharmacophore in the fight against tuberculosis.

Tuberculosis (TB) remains a global health crisis, further exacerbated by the slow pace of new treatment options, and the emergence of extreme and total drug resistance to existing drugs. The challenge to developing new antibacterial compounds with activity against Mycobacterium tuberculosis (Mtb), the causative agent of TB, is in part due to unique features of this pathogen, especially the composition and structure of its complex cell envelope. Therefore, targeting enzymes involved in cell envelope synthesis has been of major interest for anti-TB drug discovery. FAAL32 is a fatty acyl-AMP ligase involved in the biosynthesis of the cell wall mycolic acids, and a potential target for drug discovery. To rapidly advance research in this area, we initiated a drug repurposing campaign and screened a collection of 1280 approved human or veterinary drugs (Prestwick Chemical Library) using a biochemical assay that reads out FAAL32 inhibition. These efforts led to the discovery of salicylanilide closantel, and some of its derivatives as inhibitors with potent in vitro activity against M. tuberculosis. These results suggest that salicylanilide represents a potentially promising pharmacophore for the conception of novel anti-tubercular candidates targeting FAAL32 that would open new targeting opportunities. Moreover, this work illustrates the value of drug repurposing campaigns to discover new leads in challenging drug discovery fields.

α-Crystallin chaperone mimetic drugs inhibit lens γ-crystallin aggregation: Potential role for cataract prevention.

Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency-approved drugs and natural compounds for their ability to suppress or worsen H2O2 and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket-binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50-150) of HγD and one spanning the "NC tail" (residues 56-61 to 168-174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved Tm of both H2O2 oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins.

Investigation of Salicylanilides as Botulinum Toxin Antagonists.

Botulinum neurotoxin serotype A (BoNT/A) is recognized by the Centers for Disease Control and Prevention (CDC) as the most potent toxin and as a Tier 1 biowarfare agent. The severity and longevity of botulism stemming from BoNT/A is of significant therapeutic concern, and early administration of antitoxin-antibody therapy is the only approved pharmaceutical treatment for botulism. Small molecule therapeutic strategies have targeted both the heavy chain (HC) and the light chain (LC) catalytic active site and α-/ß-exosites. The LC translocation mechanism has also been studied, but an effective, nontoxic inhibitor remains underexplored. In this work, we screened a library of salicylanilides as potential translocation inhibitors. Potential leads following a primary screen were further scrutinized to identify sal30, which has a cellular minimal concentration of a drug that is required for 50% inhibition (IC50) value of 141 nM. The inquiry of salicylanilide sal30's mechanism of action was explored through a self-quenched fluorogenic substrate conjugated to bovine serum albumin (DQ-BSA) fluorescence, confocal microscopy, and vacuolar H+-ATPase (V-ATPase) inhibition assays. The summation of these findings imply that endolysosomal proton translocation through the protonophore mechanism of sal30 causes endosome pH to increase, which in turn prevents LC translocation into cytosol, a process that requires an acidic pH. Thus, the inhibition of BoNT/A activity by salicylanilides likely occurs through disruption of pH-dependent endosomal LC translocation. We further probed BoNT inhibition by sal30 using additivity analysis studies with bafilomycin A1, a known BoNT/A LC translocation inhibitor, which indicated the absence of synergy between the two ionophores.

Photoaffinity labeling of benzophenone-containing salicylanilide compounds to give an insight into the mechanism in disrupting peptidoglycan formation.

A series of salicylanilide compounds was previously identified as antibacterial agents that inhibit the peptidoglycan formation. To find the exact binding mode, we synthesized a benzophenone-containing salicylanilide compound (1) and used it as a photoaffinity probe to label Acinetobacter baumannii penicillin-binding protein (PBP1b). After incubation and photo-irradiation, the labeled protein was subjected to trypsin digestion, dialysis enrichment, LC-ESI-MS/MS analysis, and Mascot search to reveal an octadecapeptide sequence 364RQLRTEYQESDLTNQGLR381 that was labeled at E372. Our molecular docking experiments suggest a hydrophobic pocket surrounded by R367 and E372 is the binding site of salicylanilide 1. The pocket lies in between the transglycosylase and transpeptidase domains, thus binding of salicylanilide 1 can block the propagation pathway to disrupt the growth of peptidoglycan chain.