From Naturally-Sourced Protease Inhibitors to New Treatments for Fungal Infections

Proteases are involved in a broad range of physiological processes, including host invasion by fungal pathogens, and enzymatic inhibition is a key molecular mechanism controlling proteolytic activity. Importantly, inhibitors from natural or synthetic sources have demonstrated applications in biochemistry, biotechnology, and biomedicine. However, the need to discover new reservoirs of these inhibitory molecules with improved efficacy and target range has been underscored by recent protease characterization related to infection and antimicrobial resistance. In this regard, naturally-sourced inhibitors show promise for application in diverse biological systems due to high stability at physiological conditions and low cytotoxicity. Moreover, natural sources (e.g., plants, invertebrates, and microbes) provide a large reservoir of undiscovered and/or uncharacterized bioactive molecules involved in host defense against predators and pathogens. In this Review, we highlight discoveries of protease inhibitors from environmental sources, propose new opportunities for assessment of antifungal activity, and discuss novel applications to combat biomedically-relevant fungal diseases with in vivo and clinical purpose.

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Fungal Infection in Cystic Fibrosis

Current Respiratory Medicine Reviews ◽

10.2174/1573398x17666210520175847 ◽

2021 ◽

Vol 17 (2) ◽

pp. 118-124

Author(s):

Amirmehdi Sarvestani ◽

Mohammad Almasian ◽

Amirhossein Nafari

Keyword(s):

Cystic Fibrosis ◽

Fungal Infection ◽

Fungal Pathogens ◽

Respiratory Infections ◽

Fungal Infections ◽

Genetic Disorder ◽

Medical Science ◽

Resistant Species ◽

Online Databases ◽

New Treatments

Background: The prevalence of fungal infections has been increasing in recent years. Cystic fibrosis (CF) is a genetic disorder that affects organs such as the intestines, liver, pancreas, and especially the lungs. Introduction: Fungal pathogens are becoming a challenge in CF. Advanced medical science is associated with longer life expectancy in some patient groups. Method: A review was conducted on studies found on online databases, including Google Scholar, PubMed, and Scopus. Internet-based searches were performed on these databases for cystic fibrosis, respiratory infections, and fungal infection profiling to identify all relevant studies published between 2010 and 2020. Result: Fungal pathogens most frequently isolated from the respiratory tract include the Aspergillus genus, the Candida genus, Scedosporium apiospermum, and the Rasamsonia genus. In cystic fibrosis, these organisms usually colonize the respiratory and intestinal tracts and cause hypersensitivity responses and invasive diseases. Conclusion: Fungus-patient interactions are complicated and depend on various factors. Moreover, the emergence of drug-resistant species is a serious health issue, and the development of new treatments is crucial.

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Structures of Pathogenic Fungal FKBP12s Reveal Possible Self-Catalysis Function

mBio ◽

10.1128/mbio.00492-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 18

Author(s):

Nam K. Tonthat ◽

Praveen Rao Juvvadi ◽

Hengshan Zhang ◽

Soo Chan Lee ◽

Ron Venters ◽

...

Keyword(s):

Active Site ◽

Fungal Pathogens ◽

Active Sites ◽

Fungal Infections ◽

Cellular Protein ◽

Prolyl Isomerase ◽

Peptidyl Prolyl Isomerase ◽

Deep Insertion ◽

The Impact

ABSTRACT Invasive fungal infections remain difficult to treat and require novel targeting strategies. The 12-kDa FK506-binding protein (FKBP12) is a ubiquitously expressed peptidyl-prolyl isomerase with considerable homology between fungal pathogens and is thus a prime candidate for future targeting efforts to generate a panfungal strategy. Despite decades of research on FKBPs, their substrates and mechanisms of action remain unclear. Here we describe structural, biochemical, and in vivo analyses of FKBP12s from the pathogenic fungi Candida albicans , Candida glabrata , and Aspergillus fumigatus . Strikingly, multiple apo A. fumigatus and C. albicans FKBP12 crystal structures revealed a symmetric, intermolecular interaction involving the deep insertion of an active-site loop proline into the active-site pocket of an adjacent subunit. Such interactions have not been observed in previous FKBP structures. This finding indicates the possibility that this is a self-substrate interaction unique to the A. fumigatus and C. albicans fungal proteins that contain this central proline. Structures obtained with the proline in the cis and trans states provide more data in support of self-catalysis. Moreover, cysteine cross-linking experiments captured the interacting dimer, supporting the idea that it forms in solution. Finally, genetic studies exploring the impact of mutations altering the central proline and an adjacent residue provide evidence that any dimeric state formed in vivo , where FKBP12 concentrations are low, is transient. Taken together, these findings suggest a unique mechanism of self-substrate regulation by fungal FKBP12s, lending further novel understanding of this protein for future drug-targeting efforts. IMPORTANCE FKBP12 is a cis-trans peptidyl-prolyl isomerase that plays key roles in cellular protein homeostasis. FKBP12s also bind the immunosuppressive drug FK506 to inhibit the phosphatase calcineurin (CaN). CaN is required for virulence of A. fumigatus , C. albicans , C. glabrata , and other deadly fungal pathogens, marking FKBP12 and CaN as potential broad-spectrum drug targets. Here we describe structures of fungal FKBP12s. Multiple apo A. fumigatus and C. albicans FKBP12 structures reveal the insertion of a proline, conspicuously conserved in these proteins, into the active sites of adjacent molecules. This suggests that these proteins might serve as their own substrates. Cysteine disulfide trapping experiments provide support for this self-interaction and hence possible intermolecular catalysis by these enzymes.

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Alexidine dihydrochloride has broad spectrum a ctivities against diverse fungal pathogens

10.1101/429944 ◽

2018 ◽

Author(s):

Zeinab Mamouei ◽

Abdullah Alqarihi ◽

Shakti Singh ◽

Shuying Xu ◽

Michael K. Mansour ◽

...

Keyword(s):

Mammalian Cell ◽

Fungal Pathogens ◽

Fungal Infections ◽

Antifungal Drugs ◽

Immunocompromised Patients ◽

Drug Resistant ◽

Cell Toxicity ◽

Diverse Range ◽

Mammalian Cell Toxicity

AbstractInvasive fungal infections due to Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, constitute a substantial threat to hospitalized, immunocompromised patients. Further, the presence of drug-recalcitrant biofilms on medical devices, and emergence of drug-resistant fungi such as Candida auris, introduce treatment challenges with current antifungal drugs. Worse, currently there is no approved drug capable of obviating preformed biofilms which increases the chance of infection relapses. Here, we screened a small molecule Prestwick Chemical Library, consisting of 1200 FDA approved off-patent drugs, against C. albicans, C. auris and A. fumigatus, to identify those that inhibit growth of all three pathogens. Inhibitors were further prioritized for their potency against other fungal pathogens, and their ability to kill preformed biofilms. Our studies identified the bis-biguanide Alexidine dihydrochloride (AXD), as a drug with the highest antifungal and anti-biofilm activity against a diverse range of fungal pathogens. Finally, AXD significantly potentiated the efficacy of fluconazole against biofilms, displayed low mammalian cell toxicity, and eradicated biofilms growing in mice central venous catheters in vivo, highlighting its potential as a pan-antifungal drug.ImportanceThe prevalence of fungal infections has seen a rise in the past decades due to advances in modern medicine leading to an expanding population of device-associated and immunocompromised patients. Furthermore, the spectrum of pathogenic fungi has changed, with the emergence of multi-drug resistant strains such as C. auris. High mortality related to fungal infections point to major limitations of current antifungal therapy, and an unmet need for new antifungal drugs. We screened a library of repurposed FDA approved inhibitors to identify compounds with activities against a diverse range of fungi, in varied phases of growth. The assays identified Alexidine dihydrochloride (AXD) to have pronounced antifungal activity including against preformed biofilms, at concentrations lower than mammalian cell toxicity. AXD potentiated the activity of fluconazole and amphotericin B against Candida biofilms in vitro, and prevented biofilm growth in vivo. Thus AXD has the potential to be developed as a pan-antifungal, anti-biofilm drug.

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The Use of Galleria mellonella Larvae to Identify Novel Antimicrobial Agents against Fungal Species of Medical Interest

Journal of Fungi ◽

10.3390/jof4030113 ◽

2018 ◽

Vol 4 (3) ◽

pp. 113 ◽

Cited By ~ 23

Author(s):

Kevin Kavanagh ◽

Gerard Sheehan

Keyword(s):

Fungal Pathogens ◽

Antimicrobial Agents ◽

Galleria Mellonella ◽

Fungal Infections ◽

Fungal Species ◽

Molecular Techniques ◽

Antifungal Drugs ◽

Immune Priming ◽

Greater Wax Moth

The immune system of insects and the innate immune response of mammals share many similarities and, as a result, insects may be used to assess the virulence of fungal pathogens and give results similar to those from mammals. Larvae of the greater wax moth Galleria mellonella are widely used in this capacity and also for assessing the toxicity and in vivo efficacy of antifungal drugs. G. mellonella larvae are easy to use, inexpensive to purchase and house, and have none of the legal/ethical restrictions that are associated with use of mammals. Larvae may be inoculated by intra-hemocoel injection or by force-feeding. Larvae can be used to assess the in vivo toxicity of antifungal drugs using a variety of cellular, proteomic, and molecular techniques. Larvae have also been used to identify the optimum combinations of antifungal drugs for use in the treatment of recalcitrant fungal infections in mammals. The introduction of foreign material into the hemocoel of larvae can induce an immune priming effect which may operate independently with the activity of the antifungal drug. Procedures to identify this effect and limit its action are required.

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Review of the Novel Echinocandin Antifungal Rezafungin: Animal Studies and Clinical Data

Journal of Fungi ◽

10.3390/jof6040192 ◽

2020 ◽

Vol 6 (4) ◽

pp. 192

Author(s):

Yanan Zhao ◽

David S. Perlin

Keyword(s):

Animal Models ◽

Fungal Pathogens ◽

Animal Studies ◽

Fungal Infections ◽

Wide Spectrum ◽

Current Status ◽

Parent Molecule ◽

Treatment And Prevention

Rezafungin is a novel echinocandin drug being developed as a first-line option for treatment and prevention of invasive fungal infections. As a result of a structural modification in its parent molecule anidulafungin, rezafungin has acquired unique chemical stability conferring prolonged pharmacokinetics, as well as an administration advantage in the clinical setting compared to other drugs in the same class. Rezafungin displays potent in vitro activity against a wide spectrum of fungal pathogens, which is reflected in robust in vivo efficacy and/or pharmacodynamic studies using various animal models as well as in promising clinical trials data. This review describes in vivo characterization of rezafungin using animal models, current status of clinical development and key findings from these studies.

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Alexidine Dihydrochloride Has Broad-Spectrum Activities against Diverse Fungal Pathogens

mSphere ◽

10.1128/msphere.00539-18 ◽

2018 ◽

Vol 3 (5) ◽

Cited By ~ 8

Author(s):

Zeinab Mamouei ◽

Abdullah Alqarihi ◽

Shakti Singh ◽

Shuying Xu ◽

Michael K. Mansour ◽

...

Keyword(s):

Mammalian Cell ◽

Fungal Pathogens ◽

Fungal Infections ◽

Antifungal Drugs ◽

Immunocompromised Patients ◽

Cell Toxicity ◽

Diverse Range ◽

Content Type ◽

Mammalian Cell Toxicity

ABSTRACT Invasive fungal infections due to Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans constitute a substantial threat to hospitalized immunocompromised patients. Further, the presence of drug-recalcitrant biofilms on medical devices and emergence of drug-resistant fungi, such as Candida auris, introduce treatment challenges with current antifungal drugs. Worse, currently there is no approved drug capable of obviating preformed biofilms, which increase the chance of infection relapses. Here, we screened a small-molecule New Prestwick Chemical Library, consisting of 1,200 FDA-approved off-patent drugs against C. albicans, C. auris, and A. fumigatus, to identify those that inhibit growth of all three pathogens. Inhibitors were further prioritized for their potency against other fungal pathogens and their ability to kill preformed biofilms. Our studies identified the bis-biguanide alexidine dihydrochloride (AXD) as a drug with the highest antifungal and antibiofilm activity against a diverse range of fungal pathogens. Finally, AXD significantly potentiated the efficacy of fluconazole against biofilms, displayed low mammalian cell toxicity, and eradicated biofilms growing in mouse central venous catheters in vivo, highlighting its potential as a pan-antifungal drug. IMPORTANCE The prevalence of fungal infections has seen a rise in the past decades due to advances in modern medicine leading to an expanding population of device-associated and immunocompromised patients. Furthermore, the spectrum of pathogenic fungi has changed, with the emergence of multidrug-resistant strains such as C. auris. High mortality related to fungal infections points to major limitations of current antifungal therapy and an unmet need for new antifungal drugs. We screened a library of repurposed FDA-approved inhibitors to identify compounds with activities against a diverse range of fungi in varied phases of growth. The assays identified alexidine dihydrochloride (AXD) to have pronounced antifungal activity, including against preformed biofilms, at concentrations lower than mammalian cell toxicity. AXD potentiated the activity of fluconazole and amphotericin B against Candida biofilms in vitro and prevented biofilm growth in vivo. Thus, AXD has the potential to be developed as a pan-antifungal, antibiofilm drug.

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Fungal infection in cystic fibrosis

Current Hypertension Reviews ◽

10.2174/1573402117666210511012609 ◽

2021 ◽

Vol 17 ◽

Author(s):

Amirmehdi Sarvestani ◽

Mohammad Almasian ◽

Amirhossein Nafari

Keyword(s):

Cystic Fibrosis ◽

Fungal Infection ◽

Fungal Pathogens ◽

Respiratory Infections ◽

Fungal Infections ◽

Genetic Disorder ◽

Medical Science ◽

Resistant Species ◽

Online Databases ◽

New Treatments

Background: The prevalence of fungal infections has been increasing in recent years. Cystic fibrosis (CF) is a genetic disorder that affects organs such as the intestines, liver, pancreas, and especially the lungs. Introduction: Fungal pathogens are becoming a challenge in CF. Advanced medical science is associated with longer life expectancy in some patient groups. Method: A review was conducted on studies found on such online databases as Google Scholar, PubMed, and Scopus. Internet-based searches were performed on these databases for cystic fibrosis, respiratory infections, and fungal infection profiling to identify all relevant studies published between 2010 and 2020. Result: Fungal pathogens most frequently isolated from the respiratory tract include the Aspergillus genus, the Candida genus, Scedosporium apiospermum, and the Rasamsonia genus. In cystic fibrosis, these organisms usually colonize the respiratory and intestinal tracts and cause hypersensitivity responses and invasive diseases. Conclusion: Fungus-patient interactions are complicated and depend on various factors. Moreover, the emergence of drug-resistant species is a serious health issue, and the development of new treatments is crucial.

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Galleria mellonella for the Evaluation of Antifungal Efficacy against Medically Important Fungi, a Narrative Review

Microorganisms ◽

10.3390/microorganisms8030390 ◽

2020 ◽

Vol 8 (3) ◽

pp. 390 ◽

Cited By ~ 8

Author(s):

Sana Jemel ◽

Jacques Guillot ◽

Kalthoum Kallel ◽

Françoise Botterel ◽

Eric Dannaoui

Keyword(s):

Fungal Pathogens ◽

Galleria Mellonella ◽

Fungal Infections ◽

Antifungal Susceptibility ◽

Antifungal Drugs ◽

New Drugs ◽

Development Of Resistance ◽

Antifungal Efficacy

The treatment of invasive fungal infections remains challenging and the emergence of new fungal pathogens as well as the development of resistance to the main antifungal drugs highlight the need for novel therapeutic strategies. Although in vitro antifungal susceptibility testing has come of age, the proper evaluation of therapeutic efficacy of current or new antifungals is dependent on the use of animal models. Mammalian models, particularly using rodents, are the cornerstone for evaluation of antifungal efficacy, but are limited by increased costs and ethical considerations. To circumvent these limitations, alternative invertebrate models, such as Galleria mellonella, have been developed. Larvae of G. mellonella have been widely used for testing virulence of fungi and more recently have proven useful for evaluation of antifungal efficacy. This model is suitable for infection by different fungal pathogens including yeasts (Candida, Cryptococcus, Trichosporon) and filamentous fungi (Aspergillus, Mucorales). Antifungal efficacy may be easily estimated by fungal burden or mortality rate in infected and treated larvae. The aim of the present review is to summarize the actual data about the use of G. mellonella for testing the in vivo efficacy of licensed antifungal drugs, new drugs, and combination therapies.

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Retinoids in Fungal Infections: From Bench to Bedside

Pharmaceuticals ◽

10.3390/ph14100962 ◽

2021 ◽

Vol 14 (10) ◽

pp. 962

Author(s):

Terenzio Cosio ◽

Roberta Gaziano ◽

Guendalina Zuccari ◽

Gaetana Costanza ◽

Sandro Grelli ◽

...

Keyword(s):

Fungal Pathogens ◽

English Language ◽

Fungal Infections ◽

In Vivo Studies ◽

Current Evidence ◽

Candida Spp ◽

Double Blind ◽

Wide Range

Retinoids—a class of chemical compounds derived from vitamin A or chemically related to it—are used especially in dermatology, oncohematology and infectious diseases. It has been shown that retinoids—from their first generation—exert a potent antimicrobial activity against a wide range of pathogens, including bacteria, fungi and viruses. In this review, we summarize current evidence on retinoids’ efficacy as antifungal agents. Studies were identified by searching electronic databases (MEDLINE, EMBASE, PubMed, Cochrane, Trials.gov) and reference lists of respective articles from 1946 to today. Only articles published in the English language were included. A total of thirty-nine articles were found according to the criteria. In this regard, to date, In vitro and In vivo studies have demonstrated the efficacy of retinoids against a broad-spectrum of human opportunistic fungal pathogens, including yeast fungi that normally colonize the skin and mucosal surfaces of humans such as Candida spp., Rhodotorula mucilaginosa and Malassezia furfur, as well as environmental moulds such as Aspergillus spp., Fonsecae monofora and many species of dermatophytes associated with fungal infections both in humans and animals. Notwithstanding a lack of double-blind clinical trials, the efficacy, tolerability and safety profile of retinoids have been demonstrated against localized and systemic fungal infections.

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1272. Efficacy of a Non-Peptide, Small Molecule Mimic of Host Defense Proteins in Mouse Models of Disseminated Candidiasis and Aspergillosis

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1456 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S653-S653

Author(s):

Richard W Scott ◽

Simon D P Baugh ◽

Katie B Freeman ◽

Damian G Weaver ◽

Anna L Chaly ◽

...

Keyword(s):

Host Defense ◽

Limit Of Detection ◽

Fungal Infections ◽

Positive Control ◽

Chemical Diversity ◽

Independent Contractor ◽

Defense Proteins ◽

Disseminated Candidiasis ◽

Low Cytotoxicity

Abstract Background We are developing a series of small nonpeptide mimics of host defense proteins (smHDPs) for antifungal applications. MICs < 0.1 µg/ ml have been demonstrated against multiple yeast and mold species. Their potential therapeutic utility has been evaluated in animal models of disseminated fungal infections. Methods Susceptibility testing against clinical isolates of Candida albicans (CA) and Aspergillus fumigatus (AF) was done according to CLSI guidelines. Cytotoxicity (CC50) in mouse 3T3 and human HepG2 cells was determined in an MTS assay (Promega Cell Titer 96). For candidiasis, neutropenic CD-1 mice were infected IV with 3.5 x 104 CFU CA and test agents were administered subcutaneously (SC) once (QD) or twice (BID) daily beginning 2 hrs post-infection (PI). The positive control was fluconazole (20 mg/kg PO QD). Kidney burdens were measured at 24 hrs PI (n = 5/grp) and survival was measured at 2 weeks after 5 days of treatment (n = 8/grp). For aspergillosis, neutropenic CD-1 mice were infected IV with 4.5 log10 spores AF and test agents were administered SC BID for 5 days beginning 24 hrs PI. The positive control was posaconazole (20 mg/kg PO QD). Liver and kidney fungal burdens were measured 24 hrs after the last dose (n = 10/grp). Results The lead smHDP, FC-5096, has MICs of 0.08 and 0.024 ug/ml, respectively, against the CA and AF clinical isolate strains used in the mouse efficacy studies. Selectivity for antifungal vs. mammalian cell cytotoxicity (MIC/CC50) was >1,000 fold. In the disseminated candidiasis model, FC-5096 significantly reduced fungal kidney burdens relative to untreated mice to levels found at treatment onset (P < 0.0001). Full survival over 2 weeks was observed after 5 days of BID treatment with FC-5096. All untreated mice succumbed between days 3 – 6 PI. Efficacy was comparable to fluconazole. In the disseminated aspergillosis model, FC-5096 produced up to 2.5 log10 reductions in fungal liver burdens and efficacy was significantly better than posaconazole (P = 0.0251). In kidney, both FC-5096 and posaconazole caused > 1.6 log10 reductions in fungal burdens that exceeded the limit of detection. Conclusion The high antifungal potency, low cytotoxicity and robust in vivo efficacy support further study of FC-5096 for clinical development. Disclosures Richard W. Scott, PhD, Fox Chase Chemical Diversity Center (Employee) Simon DP Baugh, PhD, Fox Chase Chemical Diversity Center (Employee) Mark E Pulse, MS, Fox Chase Chemical Diversity Center (Independent Contractor)

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