Authors: Daniel L Hurdiss, Priscila El Kazzi, Lisa Bauer...

Enteroviruses are globally prevalent human pathogens responsible for many diseases. The nonstructural protein 2C is a AAA+ helicase and plays a key role in enterovirus replication. Drug repurposing screens identified 2C-targeting compounds such as fluoxetine and dibucaine, but how they inhibit 2C is unknown. Here, we present a crystal structure of the soluble and monomeric fragment of coxsackievirus B3 2C protein in complex with ()-fluoxetine (SFX), revealing an allosteric binding site. To study the functional consequences of SFX binding, we engineered an adenosine triphosphatase (ATPase)–competent, hexameric 2C protein. Using this system, we show that SFX, dibucaine, HBB [2-(α-hydroxybenzyl)-benzimidazole], and guanidine hydrochloride inhibit 2C ATPase activity. Moreover, cryo–electron microscopy analysis demonstrated that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition. Collectively, these results provide important insights into 2C inhibition and a robust engineering strategy for structural, functional, and drug-screening analysis of 2C proteins.

PubMed: 34985963
DOI: 10.1126/sciadv.abj7615

Authors: Rafeh Oualha, Yosser Zina Abdelkrim, Ikram Guizani...

Drug repurposing is a promising approach towards the discovery of novel treatments against Neglected Tropical Diseases, such as Leishmaniases, presenting the advantage of reducing both costs and duration of the drug discovery process. In previous work, our group developed a Machine Learning pipeline for the repurposing of FDA-approved drugs against parasites. The present study is focused on an validation of this approach by assessing the antileishmanial effects of 10 predicted drug candidates. First, we evaluated the drugs' activity against promastigotes from two strains of and one of , which caused distinct clinical manifestations, using an MTT assay. The standard anti- drug Amphotericin B was used as a positive control. Five molecules demonstrated anti- effects, out of which Acebutolol, Prilocaine and Phenylephrine are described herein for the first time. When tested on promastigote growth, Acebutolol displayed IC values ranging from 69.28 to 145.53 µg/mL. Prilocaine exhibited IC values between 33.10 and 45.81 µg/mL. Phenylephrine, on the other hand, presented IC values >200 µg/mL. The two remaining drugs, Dibucaine and Domperidone, exhibited significantly low IC values varying between 0.58 and 1.05 µg/mL, and 6.30 and 8.17 µg/mL, respectively. Both compounds were previously described as anti- agents . All five compounds demonstrated no notable cytotoxic effects on THP-1-derived macrophages at the IC concentrations, allowing for their testing on the intracellular form of and parasites. Interestingly, all compounds exhibited antileishmanial activity on amastigotes with enhanced IC values compared to the corresponding promastigotes. Noticeably, Dibucaine and Domperidone displayed IC values of at most 1.99 µg/mL. Acebutolol, Prilocaine and Phenylephrine showed IC values ranging from 13.84 to 66.81 µg/mL. Our previously published Computer-Aided repositioning pipelines of FDA-approved drugs as antileishmanial agents identified Dibucaine and Domperidone as candidates in support of previous studies. This study consolidates such findings through the validation against 2 species, highly prevalent in Africa and Middle East, and reveals Acebutolol, Prilocaine, and Phenylephrine as novel anti- effectors, confirming the relevance of our approach and calling for further investigations.

Topics: Drug Repositioning; Machine Learning; Antiprotozoal Agents; Humans; Inhibitory Concentration 50; Leishmania infantum; Leishmania major; Leishmaniasis; Leishmania; Drug Discovery

PubMed: 39391884
DOI: 10.3389/fcimb.2024.1403589

Review

Authors: Tyler A Kalajian, Joseph A Cannella, Ananya Vasudevan...

Over-the-counter (OTC) local anesthetics have historically been used to alleviate pain in several common conditions including toothache and sore throat. With a rise in chronic conditions and an aging population, there has been an increase in associated chronic pain-related disorders. Individuals with chronic pain often seek OTC treatments for quick and accessible pain relief. There are several common OTC local anesthetics, including benzocaine, lidocaine, and dibucaine, which are readily available to patients in several formulations. In order to appropriately advise patients on the use of local anesthetics, it is important to understand their key characteristics, including the mechanism of action, clinical properties, pharmacokinetics, clinical applications, and adverse reactions, which may occur.

Topics: Humans; Aged; Anesthetics, Local; Chronic Pain; Lidocaine; Benzocaine; Dibucaine; Anesthetics

PubMed: 37750534
DOI: 10.1111/papr.13298

Authors: Malavika Prithviraj, Takehiro Kado, Jacob A Mayfield...

The intracellular membrane domain (IMD) is a laterally discrete region of the mycobacterial plasma membrane, enriched in the subpolar region of the rod-shaped cell. Here, we report genome-wide transposon sequencing to discover the controllers of membrane compartmentalization in Mycobacterium smegmatis. The putative gene showed the most significant effect on recovery from membrane compartment disruption by dibucaine. Enzymatic analysis of Cfa and lipidomic analysis of a deletion mutant (Δ) demonstrated that Cfa is an essential methyltransferase for the synthesis of major membrane phospholipids containing a C monomethyl-branched stearic acid, also known as tuberculostearic acid (TBSA). TBSA has been intensively studied due to its abundant and genus-specific production in mycobacteria, but its biosynthetic enzymes had remained elusive. Cfa catalyzed the -adenosyl-l-methionine-dependent methyltransferase reaction using oleic acid-containing lipid as a substrate, and Δ accumulated C oleic acid, suggesting that Cfa commits oleic acid to TBSA biosynthesis, likely contributing directly to lateral membrane partitioning. Consistent with this model, Δ displayed delayed restoration of subpolar IMD and delayed outgrowth after bacteriostatic dibucaine treatment. These results reveal the physiological significance of TBSA in controlling lateral membrane partitioning in mycobacteria. As its common name implies, tuberculostearic acid is an abundant and genus-specific branched-chain fatty acid in mycobacterial membranes. This fatty acid, 10-methyl octadecanoic acid, has been an intense focus of research, particularly as a diagnostic marker for tuberculosis. It was discovered in 1934, and yet the enzymes that mediate the biosynthesis of this fatty acid and the functions of this unusual fatty acid in cells have remained elusive. Through a genome-wide transposon sequencing screen, enzyme assay, and global lipidomic analysis, we show that Cfa is the long-sought enzyme that is specifically involved in the first step of generating tuberculostearic acid. By characterizing a deletion mutant, we further demonstrate that tuberculostearic acid actively regulates lateral membrane heterogeneity in mycobacteria. These findings indicate the role of branched fatty acids in controlling the functions of the plasma membrane, a critical barrier for the pathogen to survive in its human host.

Topics: Humans; Dibucaine; Mycobacterium; Stearic Acids; Fatty Acids; Oleic Acid; Methyltransferases

PubMed: 36976029
DOI: 10.1128/mbio.03396-22

Authors: Wei Yu, Lin Wang, Wei-Ying Ren...

Recent evidence shows a close link between Parkinson's disease (PD) and cardiac dysfunction with limited treatment options. Mitophagy plays a crucial role in the control of mitochondrial quantity, metabolic reprogramming and cell differentiation. Mutation of the mitophagy protein Parkin is directly associated with the onset of PD. Parkin-independent receptor-mediated mitophagy is also documented such as BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and FUN14 domain containing 1 (FUNDC1) for receptor-mediated mitophagy. In this study we investigated cardiac function and mitophagy including FUNDC1 in PD patients and mouse models, and evaluated the therapeutic potential of a SGLT2 inhibitor empagliflozin. MPTP-induced PD model was established. PD patients and MPTP mice not only displayed pronounced motor defects, but also low plasma FUNDC1 levels, as well as cardiac ultrastructural and geometric anomalies (cardiac atrophy, interstitial fibrosis), functional anomalies (reduced E/A ratio, fractional shortening, ejection fraction, cardiomyocyte contraction) and mitochondrial injury (ultrastructural damage, UCP2, PGC1α, elevated mitochondrial Ca uptake proteins MCU and VDAC1, and mitochondrial apoptotic protein calpain), dampened autophagy, FUNDC1 mitophagy and apoptosis. By Gene set enrichment analysis (GSEA), we found overtly altered glucose transmembrane transport in the midbrains of MPTP-treated mice. Intriguingly, administration of SGLT2 inhibitor empagliflozin (10 mg/kg, i.p., twice per week for 2 weeks) in MPTP-treated mice significantly ameliorated myocardial anomalies (with exception of VDAC1), but did not reconcile the motor defects or plasma FUNDC1. FUNDC1 global knockout (FUNDC1 mice) did not elicit any phenotype on cardiac geometry or function in the absence or presence of MPTP insult, but it nullified empagliflozin-caused cardioprotection against MPTP-induced cardiac anomalies including remodeling (atrophy and fibrosis), contractile dysfunction, Ca homeostasis, mitochondrial (including MCU, mitochondrial Ca overload, calpain, PARP1) and apoptotic anomalies. In neonatal and adult cardiomyocytes, treatment with PD neurotoxin preformed fibrils of α-synuclein (PFF) caused cytochrome c release and cardiomyocyte mechanical defects. These effects were mitigated by empagliflozin (10 μM) or MCU inhibitor Ru360 (10 μM). MCU activator kaempferol (10 μM) or calpain activator dibucaine (500 μM) nullified the empagliflozin-induced beneficial effects. These results suggest that empagliflozin protects against PD-induced cardiac anomalies, likely through FUNDC1-mediated regulation of mitochondrial integrity.

Topics: Adult; Humans; Mice; Animals; Sodium-Glucose Transporter 2 Inhibitors; Parkinson Disease; Calpain; Ventricular Remodeling; Mitochondrial Proteins; Ubiquitin-Protein Ligases; Atrophy; Fibrosis; Membrane Proteins

PubMed: 37679644
DOI: 10.1038/s41401-023-01144-0

Review

Authors: Chunhui Yin, Haomiao Zhao, Xiaoyi Xia...

Picornaviruses, which are positive-stranded, non-enveloped RNA viruses, are known to infect people and animals with a broad spectrum of diseases. Among the nonstructural proteins in picornaviruses, 2C proteins are highly conserved and exhibit multiple structural domains, including amphipathic α-helices, an ATPase structural domain, and a zinc finger structural domain. This review offers a comprehensive overview of the functional structures of picornaviruses' 2C protein. We summarize the mechanisms by which the 2C protein enhances viral replication. 2C protein interacts with various host factors to form the replication complex, ultimately promoting viral replication. We review the mechanisms through which picornaviruses' 2C proteins interact with the NF-κB, RIG-I, MDA5, NOD2, and IFN pathways, contributing to the evasion of the antiviral innate immune response. Additionally, we provide an overview of broad-spectrum antiviral drugs for treating various enterovirus infections, such as guanidine hydrochloride, fluoxetine, and dibucaine derivatives. These drugs may exert their inhibitory effects on viral infections by targeting interactions with 2C proteins. The review underscores the need for further research to elucidate the precise mechanisms of action of 2C proteins and to identify additional host factors for potential therapeutic intervention. Overall, this review contributes to a deeper understanding of picornaviruses and offers insights into the antiviral strategies against these significant viral pathogens.

Topics: Humans; Animals; Picornaviridae; NF-kappa B; RNA; Virus Replication; Antiviral Agents; Structure-Activity Relationship

PubMed: 38465233
DOI: 10.3389/fcimb.2024.1347615

Authors: Chi Zhang, Jorge D Calderin, Logan R Hurst...

Sphingolipids are essential in membrane trafficking and cellular homeostasis. Here, we show that sphingolipids containing very long-chain fatty acids (VLCFAs) promote homotypic vacuolar fusion in Saccharomyces cerevisiae. The elongase Elo3 adds the last two carbons to VLCFAs that are incorporated into sphingolipids. Cells lacking Elo3 have fragmented vacuoles, which is also seen when WT cells are treated with the sphingolipid synthesis inhibitor Aureobasidin-A. Isolated elo3Δ vacuoles show acidification defects and increased membrane fluidity, and this correlates with deficient fusion. Fusion arrest occurs at the tethering stage as elo3Δ vacuoles fail to cluster efficiently in vitro. Unlike HOPS and fusogenic lipids, GFP-Ypt7 does not enrich at elo3Δ vertex microdomains, a hallmark of vacuole docking prior to fusion. Pulldown assays using bacterially expressed GST-Ypt7 showed that HOPS from elo3Δ vacuole extracts failed to bind GST-Ypt7 while HOPS from WT extracts interacted strongly with GST-Ypt7. Treatment of WT vacuoles with the fluidizing anesthetic dibucaine recapitulates the elo3Δ phenotype and shows increased membrane fluidity, mislocalized GFP-Ypt7, inhibited fusion, and attenuated acidification. Together these data suggest that sphingolipids contribute to Rab-mediated tethering and docking required for vacuole fusion.

Topics: Vacuoles; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sphingolipids; Membrane Fusion; rab GTP-Binding Proteins; Fatty Acids; Acetyltransferases

PubMed: 39307308
DOI: 10.1016/j.jbc.2024.107808

Novel treatment and insight for irradiation-induced injuries: Dibucaine ameliorates irradiation-induced testicular injury by inhibiting fatty acid oxidation in primary Leydig cells.

Authors: Lingxiang Ran, Qiu Chen, Xingyu Lu...

BACKGROUND

Male infertility is a worldwide problem but few treatments, especially irradiation-induced testicular injury. The aim of this research was to investigate novel drugs for the treatment of irradiation-induced testicular injury.

METHODS

We administered dibucaine (0.8 mg/kg) intraperitoneally to male mice (6 mice per group) after five consecutive daily 0.5 Gy whole-body irradiation, and evaluated its ameliorating efficacy by testicular HE staining and morphological measurements. Drug affinity responsive target stability assay (Darts) were used to find target protein and pathway; mouse primary Leydig cells were isolated and to explore the mechanism (Flow cytometry, Western blot, and Seahorse palmitate oxidative stress assays); finally rescue experiments were completed by combining dibucaine with fatty acid oxidative pathway inhibitors and activators.

RESULTS

The testicular HE staining and morphological measurements in dibucaine treatment group was significantly better than that in irradiation group (P < 0.05); sperm motility and mRNA levels of spermatogenic cell markers were also higher than those in the latter (P < 0.05). Darts and Western blot results showed that dibucaine targets CPT1A and downregulate fatty acid oxidation. Flow cytometry, Western blot, and Palmitate oxidative stress assays of primary Leydig cells demonstrated that dibucaine inhibits fatty acid oxidation in Leydig cells. Dibucaine combined with etomoxir/baicalin confirmed that its inhibition of fatty acid oxidation was beneficial in ameliorating irradiation-induced testicular injury.

CONCLUSIONS

In conclusion, our data suggest that dibucaine ameliorates irradiation-induced testicular injury in mice by inhibiting fatty acid oxidation in Leydig cells. This will provide novel ideas for the treatment of irradiation-induced testicular injury.

Topics: Humans; Male; Mice; Animals; Leydig Cells; Dibucaine; Sperm Motility; Testis; Testicular Diseases; Fatty Acids; Palmitates

PubMed: 37224756
DOI: 10.1016/j.biopha.2023.114903