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International Journal of Molecular... Oct 2023N-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...
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 IC 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.
Topics: Rats; Animals; Receptors, N-Methyl-D-Aspartate; Pentamidine; Guanidines; Hippocampus; Cells, Cultured; N-Methylaspartate
PubMed: 37958669
DOI: 10.3390/ijms242115685 -
Human Molecular Genetics Feb 2024GRID1 and GRID2 encode the enigmatic GluD1 and GluD2 proteins, which form tetrameric receptors that play important roles in synapse organization and development of the...
GRID1 and GRID2 encode the enigmatic GluD1 and GluD2 proteins, which form tetrameric receptors that play important roles in synapse organization and development of the central nervous system. Variation in these genes has been implicated in neurodevelopmental phenotypes. We evaluated GRID1 and GRID2 human variants from the literature, ClinVar, and clinical laboratories and found that many of these variants reside in intolerant domains, including the amino terminal domain of both GRID1 and GRID2. Other conserved regions, such as the M3 transmembrane domain, show different intolerance between GRID1 and GRID2. We introduced these variants into GluD1 and GluD2 cDNA and performed electrophysiological and biochemical assays to investigate the mechanisms of dysfunction of GRID1/2 variants. One variant in the GRID1 distal amino terminal domain resides at a position predicted to interact with Cbln2/Cbln4, and the variant disrupts complex formation between GluD1 and Cbln2, which could perturb its role in synapse organization. We also discovered that, like the lurcher mutation (GluD2-A654T), other rare variants in the GRID2 M3 domain create constitutively active receptors that share similar pathogenic phenotypes. We also found that the SCHEMA schizophrenia M3 variant GluD1-A650T produced constitutively active receptors. We tested a variety of compounds for their ability to inhibit constitutive currents of GluD receptor variants and found that pentamidine potently inhibited GluD2-T649A constitutive channels (IC50 50 nM). These results identify regions of intolerance to variation in the GRID genes, illustrate the functional consequences of GRID1 and GRID2 variants, and suggest how these receptors function normally and in disease.
Topics: Humans; Central Nervous System; Mutation; Protein Domains; Receptors, Glutamate
PubMed: 37944084
DOI: 10.1093/hmg/ddad188 -
International Journal of Molecular... Sep 2023Combining pentamidine with Gram-positive-targeting antibiotics has been proven to be a promising strategy for treating infections from Gram-negative bacteria (GNB)....
Combining pentamidine with Gram-positive-targeting antibiotics has been proven to be a promising strategy for treating infections from Gram-negative bacteria (GNB). However, which antibiotics pentamidine can and cannot synergize with and the reasons for the differences are unclear. This study aimed to identify the possible mechanisms for the differences in the synergy of pentamidine with rifampicin, linezolid, tetracycline, erythromycin, and vancomycin against GNB. Checkerboard assays were used to detect the synergy of pentamidine and the different antibiotics. To determine the mechanism of pentamidine, fluorescent labeling assays were used to measure membrane permeability, membrane potential, efflux pump activity, and reactive oxygen species (ROS); the LPS neutralization assay was used to evaluate the target site; and quantitative PCR was used to measure changes in efflux pump gene expression. Our results revealed that pentamidine strongly synergized with rifampicin, linezolid, and tetracycline and moderately synergized with erythromycin, but did not synergize with vancomycin against , , , and . Pentamidine increased the outer membrane permeability but did not demolish the outer and inner membranes, which exclusively permits the passage of hydrophobic, small-molecule antibiotics while hindering the entry of hydrophilic, large-molecule vancomycin. It dissipated the membrane proton motive force and inactivated the efflux pump, allowing the intracellular accumulation of antimicrobials that function as substrates of the efflux pump, such as linezolid. These processes resulted in metabolic perturbation and ROS production which ultimately was able to destroy the bacteria. These mechanisms of action of pentamidine on GNB indicate that it is prone to potentiating hydrophobic, small-molecule antibiotics, such as rifampicin, linezolid, and tetracycline, but not hydrophilic, large-molecule antibiotics like vancomycin against GNB. Collectively, our results highlight the importance of the physicochemical properties of antibiotics and the specific mechanisms of action of pentamidine for the synergy of pentamidine-antibiotic combinations. Pentamidine engages in various pathways in its interactions with GNB, but these mechanisms determine its specific synergistic effects with certain antibiotics against GNB. Pentamidine is a promising adjuvant, and we can optimize drug compatibility by considering its functional mechanisms.
Topics: Linezolid; Vancomycin; Rifampin; Pentamidine; Escherichia coli; Reactive Oxygen Species; Anti-Bacterial Agents; Gram-Negative Bacteria; Tetracycline; Erythromycin
PubMed: 37762115
DOI: 10.3390/ijms241813812 -
Antibiotics (Basel, Switzerland) Jul 2023The aim of this work was to (i) evaluate the efficacy of a combination treatment of pentamidine with ciprofloxacin against larvae infected with an MDR strain of and...
Combination Therapy with Ciprofloxacin and Pentamidine against Multidrug-Resistant : Assessment of In Vitro and In Vivo Efficacy and the Role of Resistance-Nodulation-Division (RND) Efflux Pumps.
The aim of this work was to (i) evaluate the efficacy of a combination treatment of pentamidine with ciprofloxacin against larvae infected with an MDR strain of and (ii) determine if pentamidine acts as an efflux-pump inhibitor. Resistant clinical isolates, mutant strains overexpressing one of three RND efflux pumps (MexAB-OprM, MexCD-OprJ, and MexEF-OprN), and a strain with the same three pumps deleted were used. MIC assays confirmed that the clinical isolates and the mutants overexpressing efflux pumps were resistant to ciprofloxacin and pentamidine. The deletion of the three efflux pumps induced sensitivity to both compounds. Exposure to pentamidine and ciprofloxacin in combination resulted in the synergistic inhibition of all resistant strains in vitro, but no synergy was observed versus the efflux-pump deletion strain. The treatment of infected larvae with the combination of pentamidine and ciprofloxacin resulted in enhanced efficacy compared with the monotherapies and significantly reduced the number of proliferating bacteria. Our measurement of efflux activity from cells revealed that pentamidine had a specific inhibitory effect on the MexCD-OprJ and MexEF-OprN efflux pumps. However, the efflux activity and membrane permeability assays revealed that pentamidine also disrupted the membrane of all cells. In conclusion, pentamidine does possess some efflux-pump inhibitory activity, in addition to a more general disruptive effect on membrane integrity that accounts for its ability to potentiate ciprofloxacin activity. Notably, the enhanced efficacy of combination therapy with pentamidine and ciprofloxacin versus MDR strains in vivo merits further investigation into its potential to treat infections via this pathogen in patients.
PubMed: 37627656
DOI: 10.3390/antibiotics12081236 -
Pathogens (Basel, Switzerland) Jun 2023Infection with and may lead to diffuse cutaneous leishmaniasis. The cure is exceptional, especially for the strange case of this lady. Case report: The patient...
Infection with and may lead to diffuse cutaneous leishmaniasis. The cure is exceptional, especially for the strange case of this lady. Case report: The patient acquired the disease in childhood and remained with lesions for over 30 years, albeit several treatments. She worsened after a pregnancy, developing disseminated lesions. Miltefosine with amphotericin B and pentamidine resulted in remission. Lesions reappeared after one year, accompanied by intra-nasal infiltration of the disease. The nasal spraying of a single ampoule of pentavalent antimoniate resulted in the sustained disappearance of the nasal symptoms and all the cutaneous lesions. After over eight years, she remains disease-free, albeit under renal replacement therapy. The high nasal mucosal antimonial concentration may explain the long-lasting cure via new MHC class I epitope-specific CD8+ cell clones against present in the nasal mucosa.
PubMed: 37513737
DOI: 10.3390/pathogens12070890 -
Tropical Medicine and Infectious Disease Jul 2023The parasitic protozoan is the etiological agent of human visceral leishmaniasis in South America, an infectious disease associated with malnutrition, anemia, and...
The parasitic protozoan is the etiological agent of human visceral leishmaniasis in South America, an infectious disease associated with malnutrition, anemia, and hepatosplenomegaly. In Brazil alone, around 2700 cases are reported each year. Treatment failure can occur as a result of drug, host, and/or parasite-related factors. Here, we isolated a species from a pediatric patient with visceral leishmaniasis that did not respond to chemotherapy, experiencing a total of nine therapeutic relapses and undergoing a splenectomy. The parasite was confirmed as after sequencing of the ribosomal DNA internal transcribed spacer, and the clinical isolate, in both promastigote and amastigote forms, was submitted to susceptibility assays with all the drugs currently used in the chemotherapy of leishmaniasis. The isolate was susceptible to meglumine antimoniate, amphotericin B, pentamidine, miltefosine, and paromomycin, similarly to another strain of this species that had previously been characterized. These findings indicate that the multiples relapses observed in this pediatric patient were not due to a decrease in the drug susceptibility of this isolate; therefore, immunophysiological aspects of the patient should be further investigated to understand the basis of treatment failure in this case.
PubMed: 37505650
DOI: 10.3390/tropicalmed8070354 -
Frontiers in Pharmacology 2023Cardiotoxicity is one of the leading causes of compound attrition during drug development. Most screening platforms aim at detecting acute cardio-electrophysiological...
Cardiotoxicity is one of the leading causes of compound attrition during drug development. Most screening platforms aim at detecting acute cardio-electrophysiological changes and drug-induced chronic functional alterations are often not studied in the early stage of drug development. Therefore, we developed an assay using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that evaluates both drug-induced acute and delayed electrophysiological and cytotoxic effects of reference compounds with clinically known cardiac outcomes. hiPSC-CMs were seeded in 48-well multielectrode array (MEA) plates and were treated with four doses of reference compounds (covering and exceeding clinical free plasma peak concentrations -fC values) and MEA recordings were conducted for 4 days. Functional-electrophysiological (field-potentials) and viability (impedance) parameters were recorded with a MEA machine. To assess this platform, we tested tyrosine-kinase inhibitors with high-cardiac risk profile (sunitinib, vandetanib and nilotinib) and low-cardiac risk (erlotinib), as well as known classic cardiac toxic drugs (doxorubicin and BMS-986094), ion-channel trafficking inhibitors (pentamidine, probucol and arsenic trioxide) and compounds without known clinical cardiotoxicity (amoxicillin, cetirizine, captopril and aspirin). By evaluating the effects of these compounds on MEA parameters, the assay was mostly able to recapitulate different drug-induced cardiotoxicities, represented by a prolongation of the field potential, changes in beating rate and presence of arrhythmic events in acute (<2 h) or delayed phase ≥24 h, and/or reduction of impedance during the delayed phase (≥24 h). Furthermore, a few reference compounds were tested in hiPSC-CMs using fluorescence- and luminescence-based plate reader assays, confirming the presence or absence of cytotoxic effects, linked to changes of the impedance parameters measured in the MEA assay. Of note, some cardiotoxic effects could not be identified at acute time points (<2 h) but were clearly detected after 24 h, reinforcing the importance of chronic drug evaluation. In conclusion, the evaluation of chronic drug-induced cardiotoxicity using a hiPSC-CMs assay can contribute to the early de-risking of compounds and help optimize the drug development process.
PubMed: 37492082
DOI: 10.3389/fphar.2023.1229960 -
Viruses May 2023Coronaviruses, including SARS-CoV-2, SARS-CoV, MERS-CoV and influenza A virus, require the host proteases to mediate viral entry into cells. Rather than targeting the...
Coronaviruses, including SARS-CoV-2, SARS-CoV, MERS-CoV and influenza A virus, require the host proteases to mediate viral entry into cells. Rather than targeting the continuously mutating viral proteins, targeting the conserved host-based entry mechanism could offer advantages. Nafamostat and camostat were discovered as covalent inhibitors of TMPRSS2 protease involved in viral entry. To circumvent their limitations, a reversible inhibitor might be required. Considering nafamostat structure and using pentamidine as a starting point, a small set of structurally diverse rigid analogues were designed and evaluated in silico to guide selection of compounds to be prepared for biological evaluation. Based on the results of in silico study, six compounds were prepared and evaluated in vitro. At the enzyme level, compounds - triggered potential TMPRSS2 inhibition with low micromolar IC concentrations, but they were less effective in cellular assays. Meanwhile, compound did not trigger potential TMPRSS2 inhibition at the enzyme level, but it showed potential cellular activity regarding inhibition of membrane fusion with a low micromolar IC value of 10.87 µM, suggesting its action could be mediated by another molecular target. Furthermore, in vitro evaluation showed that compound inhibited pseudovirus entry as well as thrombin and factor Xa. Together, this study presents compound as a hit compound that might serve as a starting point for developing potential viral entry inhibitors with possible application against coronaviruses.
Topics: Humans; COVID-19; SARS-CoV-2; Benzamidines; Middle East Respiratory Syndrome Coronavirus; Virus Internalization; Antiviral Agents
PubMed: 37243257
DOI: 10.3390/v15051171 -
Pathogens (Basel, Switzerland) May 2023Leishmaniasis, a category 1 neglected protozoan disease caused by a kinetoplastid pathogen called is transmitted through dipteran insect vectors (phlebotomine, sand... (Review)
Review
Leishmaniasis, a category 1 neglected protozoan disease caused by a kinetoplastid pathogen called is transmitted through dipteran insect vectors (phlebotomine, sand flies) in three main clinical forms: fatal visceral leishmaniasis, self-healing cutaneous leishmaniasis, and mucocutaneous leishmaniasis. Generic pentavalent antimonials have long been the drug of choice against leishmaniasis; however, their success is plagued with limitations such as drug resistance and severe side effects, which makes them redundant as frontline therapy for endemic visceral leishmaniasis. Alternative therapeutic regimens based on amphotericin B, miltefosine, and paromomycin have also been approved. Due to the unavailability of human vaccines, first-line chemotherapies such as pentavalent antimonials, pentamidine, and amphotericin B are the only options to treat infected individuals. The higher toxicity, adverse effects, and perceived cost of these pharmaceutics, coupled with the emergence of parasite resistance and disease relapse, makes it urgent to identify new, rationalized drug targets for the improvement in disease management and palliative care for patients. This has become an emergent need and more relevant due to the lack of information on validated molecular resistance markers for the monitoring and surveillance of changes in drug sensitivity and resistance. The present study reviewed the recent advances in chemotherapeutic regimens by targeting novel drugs using several strategies including bioinformatics to gain new insight into leishmaniasis. has unique enzymes and biochemical pathways that are distinct from those of its mammalian hosts. In light of the limited number of available antileishmanial drugs, the identification of novel drug targets and studying the molecular and cellular aspects of these drugs in the parasite and its host is critical to design specific inhibitors targeting and controlling the parasite. The biochemical characterization of unique -specific enzymes can be used as tools to read through possible drug targets. In this review, we discuss relevant metabolic pathways and novel drugs that are unique, essential, and linked to the survival of the parasite based on bioinformatics and cellular and biochemical analyses.
PubMed: 37242374
DOI: 10.3390/pathogens12050706 -
Tropical Medicine and Infectious Disease May 2023With more than 12 million cases worldwide, leishmaniasis is one of the top 10 neglected tropical diseases. According to the WHO, there are approximately 2 million new... (Review)
Review
With more than 12 million cases worldwide, leishmaniasis is one of the top 10 neglected tropical diseases. According to the WHO, there are approximately 2 million new cases each year in foci in around 90 countries, of which 1.5 million are cutaneous leishmaniasis (CL). Cutaneous leishmaniasis (CL) is a complex cutaneous condition that is caused by a variety of species, including () , () () , () () , and () . The disease imposes a significant burden on those who are affected since it typically results in disfiguring scars and extreme social stigma. There are no vaccines or preventive treatments available, and chemotherapeutic medications, including antimonials, amphotericin B, miltefosine, paromomycin, pentamidine, and antifungal medications, have a high price tag, a significant risk of developing drug resistance, and a variety of systemic toxicities. To work around these limitations, researchers are continuously looking for brand-new medications and other forms of therapy. To avoid toxicity with systemic medication use, high cure rates have been observed using local therapy techniques such as cryotherapy, photodynamic therapy, and thermotherapy, in addition to some forms of traditional therapies, including leech and cauterization therapies. These CL therapeutic strategies are emphasized and assessed in this review to help with the process of locating the appropriate species-specific medicines with fewer side effects, lower costs, and elevated cure rates.
PubMed: 37235323
DOI: 10.3390/tropicalmed8050275