Macrocyclic inhibitors have emerged as a privileged scaffold in medicinal chemistry, offering enhanced selectivity, stability, and pharmacokinetic profiles compared to their linear counterparts. Here, we describe a novel, on-resin macrocyclization strategy for the synthesis of potent inhibitors targeting the secreted protease Major Aspartyl Peptidase 1 in Cryptococcus neoformans, a pathogen responsible for life-threatening fungal infections. By employing diverse aliphatic linkers and statine-based transition-state mimics, we constructed a focused library of 624 macrocyclic compounds. Screening identified several subnanomolar inhibitors with desirable pharmacokinetic and antifungal properties. Lead compound 25 exhibited a Ki of 180 pM, significant selectivity against host proteases, and potent antifungal activity in culture. The streamlined synthetic approach not only yielded drug-like macrocycles with potential in antifungal therapy but also provided insights into structure-activity relationships that can inform broader applications of macrocyclization in drug discovery.
- MeSH
- antifungální látky * farmakologie chemie chemická syntéza farmakokinetika MeSH
- Cryptococcus neoformans * účinky léků enzymologie MeSH
- inhibitory proteas * farmakologie chemie chemická syntéza farmakokinetika MeSH
- lidé MeSH
- makrocyklické sloučeniny * farmakologie chemie chemická syntéza farmakokinetika MeSH
- mikrobiální testy citlivosti MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antifungální látky * MeSH
- inhibitory proteas * MeSH
- makrocyklické sloučeniny * MeSH
Cryptococcosis is an invasive mycosis caused mainly by Cryptococcus gattii and C. neoformans and is treated with amphotericin B (AMB), fluconazole and 5-fluorocytosine. However, antifungal resistance, limited and toxic antifungal arsenal stimulate the search for therapeutic strategies such as drug repurposing. Among the repurposed drugs studied, the selective serotonin reuptake inhibitors (SSRIs) have shown activity against Cryptococcus spp. However, little is known about the antifungal effect of duloxetine hydrochloride (DH), a selective serotonin and norepinephrine reuptake inhibitor (SSNRI), against C. neoformans and C. gattii. In this study, DH inhibited the growth of several C. neoformans and C. gattii strains at concentrations ranging from 15.62 to 62.50 µg/mL. In addition, DH exhibited fungicidal activity ranging from 15.62 to 250 µg/mL. In biofilm, DH treatment reduced Cryptococcus spp. biomass at a level comparable to AMB, with a significant reduction (85%) for C. neoformans biofilms. The metabolic activity of C. neoformans and C. gattii biofilms decreased significantly (99%) after treatment with DH. Scanning electron micrographs confirmed the anti-biofilm activity of DH, as isolated cells could be observed after treatment. In conclusion, DH showed promising antifungal activity against planktonic cells and biofilms of C. neoformans and C. gattii, opening perspectives for further studies with DH in vivo.
- Klíčová slova
- Cryptococcus spp, Biofilm, Drug repurposing, Duloxetine hydrochloride,
- MeSH
- antifungální látky * farmakologie MeSH
- biofilmy * účinky léků MeSH
- Cryptococcus gattii * účinky léků MeSH
- Cryptococcus neoformans * účinky léků růst a vývoj MeSH
- duloxetinum hydrochlorid * farmakologie MeSH
- kryptokokóza farmakoterapie mikrobiologie MeSH
- mikrobiální testy citlivosti * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antifungální látky * MeSH
- duloxetinum hydrochlorid * MeSH
Cryptococcus neoformans is an encapsulated yeast that can cause cryptococcosis and cryptococcal meningitis, which conventional treatment involves antifungal drugs such as polyenes, flucytosine, azoles, and their combinations. However, the high cost, toxicity, and increase in fungi resistance to antifungal agents stimulate the search for therapeutic strategies such as drug repurposing and combination therapy. This study evaluated the activity of the antihypertensive verapamil (VEH) alone and combined with amphotericin B (AmB) against C. neoformans. VEH exhibited antifungal activity against C. neoformans with minimum inhibitory concentration and minimum fungicidal concentration of 118 µg per mL. The combination of VEH and AmB exhibited synergism, reducing at least eightfold both drugs' concentrations. Moreover, the combination decreased the size and glucuronoxylomannnan content of C. neoformans capsule. However, no difference was observed in ergosterol levels of C. neoformans after treatment with VEH and AmB in combination. Altogether, VEH in combination with AmB exhibits potential as a candidate as for the development of anti-cryptococcal drug.
- Klíčová slova
- Antifungal, Capsule, Cryptococcus neoformans, Synergism, Verapamil,
- MeSH
- amfotericin B farmakologie terapeutické užití MeSH
- antifungální látky farmakologie terapeutické užití MeSH
- Cryptococcus neoformans * MeSH
- flucytosin farmakologie terapeutické užití MeSH
- kryptokokóza * farmakoterapie mikrobiologie MeSH
- mikrobiální testy citlivosti MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- amfotericin B MeSH
- antifungální látky MeSH
- flucytosin MeSH
Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life-threatening invasive fungal infections. As its prevalence and drug resistance continue to rise, cryptococcosis requires new treatment options. Tapping into the potential antifungal effects of traditional drugs or combination therapy has become one of the options. This study was the first to examine the interaction of hydroxychloroquine (HCQ) and itraconazole (ITR) on Cryptococcus neoformans in vitro and in vivo. Our results showed that HCQ alone and in combination with ITR exhibited antifungal activity against C. neoformans planktonic cells. When HCQ was combined with ITR, the minimal inhibitory concentration (MIC) value of HCQ decreased to 32 μg/mL, and the MIC value of ITR decreased from 0.25 μg/mL to 0.06-0.25 μg/mL. The time-killing curve showed that the combined application of HCQ and ITR significantly shortened the killing time, dynamically defining the antifungal activity. The minimum biofilm clearance concentration (MBEC) of HCQ was only 32 μg/mL, which was significantly lower than the MIC of HCQ for planktonic cells. When combined with ITR, the MBEC of ITR decreased from 128 μg/mL to 2-1 μg/mL, and the MBEC of HCQ decreased from 32 μg/mL to 4 μg/mL, indicating a synergistic antifungal biofilm effect. In comparison to ITR alone, the combination of HCQ and ITR treatment increased the survival of C. neoformans-infected Galleria mellonella larvae and decreased the fungal burden of infected larvae. Mechanistic investigations revealed that HCQ might damage C. neoformans cell membranes, impact the structure of fungal cells, cause extracellular material leakage, and have a potent affinity for attaching to the C. neoformans genomic DNA. In conclusion, HCQ has potential clinical application in the treatment of cryptococcosis.
- Klíčová slova
- Antifungal activity, Cryptococcus neoformans, Hydroxychloroquine, Itraconazole, Synergism,
- MeSH
- antifungální látky farmakologie terapeutické užití MeSH
- Cryptococcus neoformans * MeSH
- hydroxychlorochin farmakologie terapeutické užití MeSH
- itrakonazol farmakologie MeSH
- kryptokokóza * farmakoterapie mikrobiologie MeSH
- mikrobiální testy citlivosti MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antifungální látky MeSH
- hydroxychlorochin MeSH
- itrakonazol MeSH
Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.
- MeSH
- antifungální látky chemie metabolismus farmakologie MeSH
- aspartátové proteasy antagonisté a inhibitory genetika metabolismus MeSH
- Cryptococcus neoformans enzymologie MeSH
- fungální proteiny antagonisté a inhibitory genetika metabolismus MeSH
- HIV-proteasa chemie metabolismus MeSH
- HIV enzymologie MeSH
- houby účinky léků MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- preklinické hodnocení léčiv MeSH
- rekombinantní proteiny biosyntéza chemie izolace a purifikace MeSH
- simulace molekulární dynamiky MeSH
- substrátová specifita MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- antifungální látky MeSH
- aspartátové proteasy MeSH
- fungální proteiny MeSH
- HIV-proteasa MeSH
- rekombinantní proteiny MeSH
Cryptococcosis is a potentially fatal fungal disease which has aggrandized with the emergence of AIDS and antifungal resistance. The currently used antifungals lack the broad-spectrum activity and result in several toxicities during long treatment regimens. Thus, the present study aims to evaluate the antifungal activity of cinnamaldehyde against Cryptococcus neoformans var. grubii, the etiological agent of the disease. Quantitative and qualitative in vitro fungal susceptibilities were carried out by minimum inhibitory concentration assay, flow cytometric analysis, and confocal microscopy. Micromorphological alterations were studied through scanning electron and light microscopies. "In vivo" antifungal efficacy of cinnamaldehyde was assessed. Cinnamaldehyde showed antifungal activity against C. neoformans in a dose-dependent manner. A concentration of 1.37 mg/mL of cinnamaldehyde was found to be inhibitory and fungicidal while the low concentration (0.68 mg/mL) was found to induce micromorphological changes and formation of giant/titan-like cells in this pathogen. The reparative activity of cinnamaldehyde and its ability to prolong the life even after the advent of cryptococcal meningitis in mice was also noticed. This study suggests potent anti-cryptococcal activity of cinnamaldehyde. Though, it has a couple of limitations like allergy and low bioavailability. However, these problems can be circumvented by developing suitable analogs of the compound. It, therefore, could be used as a therapeutic option against cryptococcosis and cryptococcal meningitis. Moreover, the evaluation of its pharmacokinetic and pharmacodynamic properties is desirable.
- Klíčová slova
- Cinnamaldehyde, Cryptococcosis, Giant cells, In vitro, In vivo,
- MeSH
- akrolein analogy a deriváty farmakologie MeSH
- analýza přežití MeSH
- antifungální látky farmakologie MeSH
- Cryptococcus neoformans účinky léků MeSH
- fungální léková rezistence účinky léků MeSH
- játra patologie MeSH
- kryptokokóza farmakoterapie mikrobiologie patologie MeSH
- mikrobiální testy citlivosti MeSH
- modely nemocí na zvířatech MeSH
- mozek patologie MeSH
- mykózy farmakoterapie MeSH
- myši MeSH
- plíce patologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- akrolein MeSH
- antifungální látky MeSH
- cinnamaldehyde MeSH Prohlížeč
URA5-RFLP is one of the most widely used genotyping methods relating to Cryptococcus neoformans and C. gattii consensus genotype nomenclature. In order to identify a molecular type, this method uses a visual comparison of digested PCR products of tested and reference strains, therefore any anomaly in RFLP patterns of studied isolates makes recognition difficult or impossible. This report describes a strain of VNIV type showing an atypical URA5-RFLP pattern as well as a group of AD hybrids displaying the same anomaly. The atypical RFLP pattern is the result of a point mutation and emergence of a new restriction site. Emergence of the allele presenting a new banding pattern may lead to misidentification using the URA5-RFLP technique; the results of this study as well as the literature data may suggest the spread of the allele in the environment.
- MeSH
- Cryptococcus neoformans klasifikace genetika MeSH
- genotyp MeSH
- geny hub genetika MeSH
- mikrobiologie životního prostředí MeSH
- mutace MeSH
- mykologické určovací techniky MeSH
- orotátfosforibosyltransferasa genetika MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- orotátfosforibosyltransferasa MeSH
Cryptococcal meningitis is a severe neurological infection caused by the yeast Cryptococcus neoformans. It often occurs as an opportunistic infection; rarely, it may be seen in healthy people as well. The most common source of the infection is inhalation of infected bird droppings. The cryptococci may persist in the lungs and nearby lymph nodes for a long time. There are no or mild clinical manifestations of the pulmonary infection. The disease often manifests only after the cryptococci penetrate into the CNS. The case report documents the development of cryptococcal meningitis in an immunocompetent patient. It was diagnosed by microscopic detection of the yeast in the cerebrospinal fluid. The finding was confirmed by detecting cryptococcal DNA in the cerebrospinal fluid and culture. Despite immediate initiation of antifungal therapy and intensive care, the patient died.
- MeSH
- Cryptococcus neoformans * MeSH
- DNA fungální mozkomíšní mok MeSH
- fatální výsledek MeSH
- imunokompetence MeSH
- kryptokoková meningitida * mozkomíšní mok diagnóza MeSH
- lidé MeSH
- lymfatické uzliny MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- Názvy látek
- DNA fungální MeSH
Certain non-steroidal anti-inflammatory drugs can inhibit fungal growth, fungal prostaglandin E2 production, and enzyme activation. This study aims to investigate the antifungal effect of nimesulide against pathogenic filamentous fungi and yeast. The experiments detailed below were also designed to investigate whether the action is dependent on E2 fungal prostaglandins. Our data showed that nimesulide exhibited potent antifungal activity, mainly against Trichophyton mentagrophytes (ATCC 9533) and Cryptococcus neoformans with MIC values of 2 and 62 μg/mL, respectively. This drug was also able to inhibit the growth of clinic isolates of filamentous fungi, such as Aspergillus fumigatus, and dermatophytes, such as T. rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, and M. gypseum, with MIC values ranging from 112 to 770 μg/mL. Our data also showed that the inhibition of fungal growth by nimesulide was mediated by a mechanism dependent on PGE2, which led to the inhibition of essential fungal enzymes. Thus, we concluded that nimesulide exerts a fungicidal effect against pathogenic filamentous fungi and yeast, involving the inhibition of fungal prostaglandins and fungal enzymes important to the fungal growth and colonization.
- MeSH
- antifungální látky farmakologie MeSH
- Arthrodermataceae účinky léků růst a vývoj metabolismus MeSH
- Aspergillus fumigatus účinky léků růst a vývoj metabolismus MeSH
- Cryptococcus neoformans účinky léků růst a vývoj metabolismus MeSH
- dinoproston antagonisté a inhibitory biosyntéza MeSH
- mikrobiální testy citlivosti MeSH
- sulfonamidy farmakologie MeSH
- Trichophyton účinky léků růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antifungální látky MeSH
- dinoproston MeSH
- nimesulide MeSH Prohlížeč
- sulfonamidy MeSH
BACKGROUND: We investigated the targeting of microtubules (MT) and F-actin cytoskeleton (AC) of the human pathogenic yeast Cryptococcus neoformans with agents for cancer therapy, in order to examine whether this yeast cytoskeleton could become a new antifungal target for the inhibition of cell division. METHODS: Cells treated with 10 cytoskeleton inhibitors in yeast extract peptone dextrose medium were investigated by phase-contrast and fluorescence microscopy, and growth inhibition was estimated by cell counts using a Bürker chamber and measuring absorbance for 6 days. RESULTS: Docetaxel, paclitaxel, vinblastine sulfate salt, cytochalasin D and chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] did not inhibit proliferation. The MT inhibitors methyl benzimidazole-2-ylcarbamate (BCM), nocodazole, thiabendazole (TBZ) and vincristine (VINC) disrupted MT and inhibited mitoses, but anucleated buds emerged on cells that increased in size, vacuolated and seemed to die after 2 days. The response of the cells to the presence of the actin inhibitor latrunculin A (LA) included the disappearance of actin patches, actin cables and actin rings; this arrested budding and cell division. However, in 3-4 days, resistant budding cells appeared in all 5 inhibitors. Disruption of the MT and AC and inhibition of cell division and budding persisted only when the MT and AC inhibitors were combined, i.e. VINC + LA, BCM + LA or TBZ + LA. CONCLUSION: The MT and AC of C. neoformans are new antifungal targets for the persistent inhibition of cell division by combined F-actin and MT inhibitors.
- MeSH
- aktiny antagonisté a inhibitory MeSH
- antifungální látky farmakologie MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- buněčné dělení účinky léků MeSH
- Cryptococcus neoformans účinky léků MeSH
- fluorescenční mikroskopie MeSH
- lidé MeSH
- mikrofilamenta účinky léků MeSH
- mikrotubuly účinky léků MeSH
- protinádorové látky farmakologie MeSH
- racionální návrh léčiv MeSH
- thiazolidiny farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aktiny MeSH
- antifungální látky MeSH
- bicyklické sloučeniny heterocyklické MeSH
- latrunculin A MeSH Prohlížeč
- protinádorové látky MeSH
- thiazolidiny MeSH
