scholarly journals Discovery of fungal-specific targets and inhibitors using chemical phenotyping of pathogenic spore germination

2021 ◽  
Author(s):  
Sébastien C. Ortiz ◽  
Mingwei Huang ◽  
Christina M. Hull
Keyword(s):  
Spore Germination ◽  
Mammalian Cells ◽  
Target Identification ◽  
Antifungal Drugs ◽  
Human Fungal Pathogen ◽  
Inhibitory Compounds ◽  
Transport Chain ◽  
Similar Chemical ◽  
Spore Population ◽  
Low Cytotoxicity

AbstractThere is a critical need for new antifungal drugs; however, the lack of available fungal-specific targets is a major hurdle in the development of antifungal therapeutics. Spore germination is a differentiation process absent in humans that could harbor uncharacterized fungal-specific targets. To capitalize on this possibility, we developed novel phenotypic assays to identify and characterize inhibitors of spore germination of the human fungal pathogen Cryptococcus. Using these assays, we carried out a high throughput screen of ~75,000 drug-like small molecules and identified and characterized 191 novel inhibitors of spore germination, many of which also inhibited yeast replication and demonstrated low cytotoxicity against mammalian cells. Using an automated, microscopy-based, quantitative germination assay (QGA), we discovered that germinating spore populations can exhibit unique phenotypes in response to chemical inhibitors. Through the characterization of these spore population dynamics in the presence of the newly identified inhibitors, we classified 6 distinct phenotypes based on differences in germination synchronicity, germination rates, and overall population behavior. Similar chemical phenotypes were induced by inhibitors that targeted the same cellular function or had shared substructures. Leveraging these features, we used QGAs to identify outliers among compounds that fell into similar structural groups and thus refined relevant structural moieties, facilitating target identification. This approach led to the identification of complex II of the electron transport chain as the putative target of a promising structural cluster of germination inhibitory compounds. These inhibitors showed high potency against Cryptococcus spore germination, while maintaining low cytotoxicity against mammalian cells, making them prime candidates for development into novel antifungal therapeutics.

Synthesis and Structure−Activity Relationship Studies of N-monosubstituted Aroylthioureas as Urease Inhibitors

2020 ◽  
Vol 16 ◽  
Author(s):  
Wei-Wei Ni ◽  
Hai-Lian Fang ◽  
Ya-Xi Ye ◽  
Wei-Yi Li ◽  
Li Liu ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Mammalian Cells ◽  
Intact Cell ◽  
Linear Regression Analysis ◽  
Positive Control ◽  
Acetohydroxamic Acid ◽  
Urease Inhibitors ◽  
Ic50 Value ◽  
Ic50 Values ◽  
Low Cytotoxicity

Background: Thiourea is a classical urease inhibitor usually as a positive control, and many N,N`-disubstituted thioureas have been determined as urease inhibitors. However, due to steric hindrance, N,N`-disubstituted thiourea motif could not bind urease as thiourea. On the contrary, N-monosubstituted thioureas with a tiny thiourea motif could theoretically bind into the active pocket as thiourea. Objective: A series of N-monosubstituted aroylthioureas were designed and synthesized for evaluation as urease inhibitors. Methods: Urease inhibition was determined by the indophenol method and IC50 values were calculated using computerized linear regression analysis of quantal log dose-probit functions. The kinetic parameters were estimated viasurface plasmon resonance (SPR) and by nonlinear regression analysis based on the mixed type inhibition model derived from Michaelis-Menten kinetics. Results: Compounds b2, b11and b19 reversibly inhibited urease with a mixed mechanism, and showed excellent potency against both cell-free urease and urease in intact cell, with IC50 values being 90-to 450-fold and 5-to 50-fold lower than the positive control acetohydroxamic acid, respectively. The most potent compound b11 showed IC50 value of 0.060 ±0.004μM against cell-free urease, which bound to urea binding site with a very low KDvalue (0.420±0.003nM) and a very long residence time (6.7 min). Compound b11was also demonstrated having very low cytotoxicity to mammalian cells. Conclusion: These results revealed that N-monosubstituted aroylthioureas clearly bind the active site of urease as expected, and represent a new class of urease inhibitors for the development of potential therapeutics against infections caused by ure-ase-containing pathogens.

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

scholarly journals Complex II subunit SDHD is critical for cell growth and metabolism, which can be partially restored with a synthetic ubiquinone analog

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Aloka B. Bandara ◽  
Joshua C. Drake ◽  
David A. Brown
Keyword(s):  
Mammalian Cells ◽  
Krebs Cycle ◽  
Atp Synthesis ◽  
Hek293 Cells ◽  
Knockout Mutant ◽  
Complex Ii ◽  
Growth Defects ◽  
Transport Chain ◽  
Mutant Cells

Abstract Background Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. Results Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. Conclusions Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.

scholarly journals Primary and Secondary Drug Screening Assays for Friedreich Ataxia

2011 ◽  
Vol 17 (3) ◽  
pp. 303-313 ◽  
Author(s):  
M. Grazia Cotticelli ◽  
Lynn Rasmussen ◽  
Nicole L. Kushner ◽  
Sara McKellip ◽  
Melinda Ingrum Sosa ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Mitochondrial Function ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Friedreich Ataxia ◽  
Krebs Cycle ◽  
C2c12 Cells ◽  
Transport Chain ◽  
Screening Assays ◽  
Yeast Mutants

Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix for the assembly of iron–sulfur clusters (ISCs), which are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain (ETC). Decreased expression of frataxin or the yeast frataxin orthologue, Yfh1p, is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. Using yeast depleted of Yfh1p, a high-throughput screening (HTS) assay was developed in which mitochondrial function was monitored by reduction of the tetrazolium dye WST-1 in a growth medium with a respiration-only carbon source. Of 101 200 compounds screened, 302 were identified that effectively rescue mitochondrial function. To confirm activities in mammalian cells and begin understanding mechanisms of action, secondary screening assays were developed using murine C2C12 cells and yeast mutants lacking specific complexes of the ETC, respectively. The compounds identified in this study have potential relevance for other neurodegenerative disorders associated with mitochondrial dysfunction, such as Parkinson disease.

scholarly journals Acylhydrazones as Antifungal Agents Targeting the Synthesis of Fungal Sphingolipids

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Cristina Lazzarini ◽  
Krupanandan Haranahalli ◽  
Robert Rieger ◽  
Hari Krishna Ananthula ◽  
Pankaj B. Desai ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Antifungal Agents ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Fungal Resistance ◽  
Content Type ◽  
Highly Active ◽  
Pharmacokinetic Properties ◽  
Pass Through

ABSTRACTThe incidence of invasive fungal infections has risen dramatically in recent decades. Current antifungal drugs are either toxic, likely to interact with other drugs, have a narrow spectrum of activity, or induce fungal resistance. Hence, there is a great need for new antifungals, possibly with novel mechanisms of action. Previously our group reported an acylhydrazone called BHBM that targeted the sphingolipid pathway and showed strong antifungal activity against several fungi. In this study, we screened 19 derivatives of BHBM. Three out of 19 derivatives were highly active againstCryptococcus neoformansin vitroand had low toxicity in mammalian cells. In particular, one of them, called D13, had a high selectivity index and showed better activity in an animal model of cryptococcosis, candidiasis, and pulmonary aspergillosis. D13 also displayed suitable pharmacokinetic properties and was able to pass through the blood-brain barrier. These results suggest that acylhydrazones are promising molecules for the research and development of new antifungal agents.

A Synergistic Antimicrobial Mechanism of GO: Why Oxidative Stress Can Inactivate E. coli

NANO ◽  
2020 ◽  
Vol 15 (04) ◽  
pp. 2050054 ◽  
Author(s):  
Xin Li ◽  
Haoqi Zhao ◽  
Shidong Wang ◽  
Weiwu Zou ◽  
Peiyan Yang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Antibacterial Activity ◽  
Mammalian Cells ◽  
Water Solubility ◽  
Direct Interaction ◽  
Medical Application ◽  
Future Application ◽  
Sem Images ◽  
E Coli ◽  
Low Cytotoxicity

Graphene oxide (GO), a 2D nanomaterial, is a promising material for medical application, thanks to its water solubility, antibacterial activity and relatively low cytotoxicity. However, many factors, such as lateral dimension, purity and surface chemistry, may influence its antibacterial activity, its exact mechanism is still unknown. In this work, E. coli was used as model bacterium to determine the antibacterial activity of well-dispersed GO which was obtained by a modified Hummer method and dialyzed to remove the salts and acid used in the oxidation process. After co-culture with GO for 2[Formula: see text]h, up to 90% E. coli cells were inactivated when GO concentration at 8[Formula: see text][Formula: see text]g/mL. The direct interaction was not detected in FE-SEM images and the results of [Formula: see text] potential showed that the interaction between GO and E. coli are repulsive[Formula: see text] Our results showed that GO can produce ROS and inactivate SOD and CAT enzymes in low concentration after co-cultured with E. coli which explained the antibacterial activity of GO in aqueous solution. Meanwhile, GO, with high purity, showed low cytotoxicity towards mammalian cells and did not cause any observable hemoglobin after co-cultured with blood cells. The data presented here prove that GO is effectively inhibit E. coli through inactivating SOD, CAT enzymes and the oxidative stress produced by ROS. Furthermore, the good biocompatibility promised its future application.

scholarly journals Whole-Genome Approach to Understanding the Mechanism of Action of a Histatin 5-Derived Peptide

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Cody B. Bullock ◽  
David S. McNabb ◽  
Inés Pinto
Keyword(s):  
Mechanism Of Action ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Content Type ◽  
Histatin 5 ◽  
Amino Acid Peptide ◽  
Killing Activity ◽  
Transport Chain ◽  
Mitochondrial Functions ◽  
Increased Sensitivity

ABSTRACT The incidence of opportunistic fungal infections that threaten immunocompromised patients, along with the limited arsenal of antifungal drugs, calls for renewed efforts to develop novel antifungal therapies. Antimicrobial peptides have garnered interest as potential therapeutics. Among naturally occurring peptides, histatin 5 is a well-characterized 24-amino-acid peptide with strong antifungal activity. Our lab has identified a smaller histatin derivative, KM29, with stronger activity against multiple Candida spp., prompting us to investigate its fungicidal mechanism. A genetic screen was developed to test the Saccharomyces cerevisiae genomewide deletion collection for mutants with increased or decreased peptide sensitivity. The goal was to identify genes that would reveal insights into the mechanism of action of KM29, to be assessed in Candida albicans. Several biological processes yielded increased sensitivity, with endosomal transport and vacuolar function appearing at high frequencies. Among the pathways involved in increased resistance, mitochondrial function showed the highest normalized genome frequency; hence, we focused on characterizing this pathway. KM29 localizes to mitochondria, and the killing activity depends on a functional electron transport chain. In addition, KM29 triggered reactive oxygen species (ROS) production, which was responsible for some cell death but insufficient to account for the complete killing activity. In agreement with this finding, we found that KM29 induced mitochondrial fragmentation and a mild loss of mitochondrial membrane potential. Furthermore, respiratory mutants exhibited severely diminished KM29 uptake. We confirmed this behavior in a C. albicans respiratory mutant. Taking our findings together, this work delineates the mitochondrial functions associated with KM29 fungicidal activity and provides additional pathways for further characterization in Candida spp.

scholarly journals In Vitro Antifungal Activities of a Series of Dication-Substituted Carbazoles, Furans, and Benzimidazoles

1998 ◽  
Vol 42 (10) ◽  
pp. 2503-2510 ◽  
Author(s):  
Maurizio Del Poeta ◽  
Wiley A. Schell ◽  
Christine C. Dykstra ◽  
Susan K. Jones ◽  
Richard R. Tidwell ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Candida Albicans ◽  
Fusarium Solani ◽  
Antifungal Agents ◽  
Antifungal Drugs ◽  
Inhibitory Compounds ◽  
Wide Range ◽  
Fluconazole Resistant ◽  
Resistant Strains

ABSTRACT Aromatic dicationic compounds possess antimicrobial activity against a wide range of eucaryotic pathogens, and in the present study an examination of the structures-functions of a series of compounds against fungi was performed. Sixty-seven dicationic molecules were screened for their inhibitory and fungicidal activities againstCandida albicans and Cryptococcus neoformans. The MICs of a large number of compounds were comparable to those of the standard antifungal drugs amphotericin B and fluconazole. Unlike fluconazole, potent inhibitory compounds in this series were found to have excellent fungicidal activities. The MIC of one of the most potent compounds against C. albicans was 0.39 μg/ml, and it was the most potent compound against C. neoformans (MIC, ≤0.09 μg/ml). Selected compounds were also found to be active againstAspergillus fumigatus, Fusarium solani,Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans. Since some of these compounds have been safely given to animals, these classes of molecules have the potential to be developed as antifungal agents.

scholarly journals Liposomes Composed by Membrane Lipid Extracts from Macrophage Cell Line as a Delivery of the Trypanocidal N,N’-Squaramide 17 towards Trypanosoma cruzi

Materials ◽  
2020 ◽  
Vol 13 (23) ◽  
pp. 5505
Author(s):  
Christian Rafael Quijia ◽  
Cínthia Caetano Bonatto ◽  
Luciano Paulino Silva ◽  
Milene Aparecida Andrade ◽  
Clenia Santos Azevedo ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Entrapment Efficiency ◽  
Membrane Lipid ◽  
Raw 264.7 Cells ◽  
Hydrodynamic Diameter ◽  
Raw 264.7 ◽  
Low Cytotoxicity

Chagas is a neglected tropical disease caused by Trypanosoma cruzi, and affects about 25 million people worldwide. N, N’-Squaramide 17 (S) is a trypanocidal compound with relevant in vivo effectiveness. Here, we produced, characterized, and evaluated cytotoxic and trypanocidal effects of macrophage-mimetic liposomes from lipids extracted of RAW 264.7 cells to release S. As results, the average hydrodynamic diameter and Zeta potential of mimetic lipid membranes containing S (MLS) was 196.5 ± 11 nm and −61.43 ± 2.3 mV, respectively. Drug entrapment efficiency was 73.35% ± 2.05%. After a 72 h treatment, MLS was observed to be active against epimastigotes in vitro (IC50 = 15.85 ± 4.82 μM) and intracellular amastigotes (IC50 = 24.92 ± 4.80 μM). Also, it induced low cytotoxicity with CC50 of 1199.50 ± 1.22 μM towards VERO cells and of 1973.97 ± 5.98 μM in RAW 264.7. MLS also induced fissures in parasite membrane with a diameter of approximately 200 nm in epimastigotes. MLS showed low cytotoxicity in mammalian cells and high trypanocidal activity revealing this nanostructure a promising candidate for the development of Chagas disease treatment.

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