Continuous dosing of nikkomycin Z against systemic candidiasis, in vivo and in vitro correlates.

BACKGROUND: Candidiasis is the most common cause of fungal sepsis, and new agents are of interest to ameliorate current deficiencies in therapy. Nikkomycin Z (NIKZ) is an inhibitor of chitin synthase, interfering with fungal cell wall development. OBJECTIVES/METHODS: We studied NIKZ therapy of disseminated murine candidiasis, via continuous drug exposure, in drinking water, to compensate for rapid clearance of the drug. RESULTS: Drinking, and thus drug intake in the NIKZ groups, as well as body weight, was affected by the degree of illness. NIKZ effect on survival, despite reduced drinking initially after infection, was highly efficacious and dose-related, and comparable to fluconazole, though neither were curative with the regimens employed. The challenge was rapidly lethal to all untreated animals, whereas NIKZ groups achieved >50% survival. Assays of residual fungal infection were consistent with impressions of efficacy based on survival. Although NIKZ MIC for Candida albicans appeared unpromising, mycelial formation assays more closely correlated with in vivo observations. CONCLUSIONS: In vitro-in vivo disparity may be explained by NIKZ tissue concentration in the target tissue and/or by enhanced NIKZ action on mycelial formation, a morphological change in vivo wherein chitin synthesis is more critical, compared to NIKZ activity in inhibiting planktonic growth. A sustained release oral form of NIKZ in drug development for humans could hold promise, possibly also in future exploring previously demonstrated synergy in vitro with other antifungals.

Synergy In Vitro of Nikkomycin Z with Azole Against the Invasive Form of Candida albicans.

In a previous study, therapeutic activity of nikkomycin Z (NZ) in a model of invasive candidiasis did not appear to correlate with lesser activity in vitro (using classical MIC methods) with planktonic organisms. However, NZ potency was much greater assaying activity in vitro against germ tubes, the initiator of the invasive mycelial form of the fungus, as occurs in infected tissues. Synergy has been demonstrated for NZ and other drugs, notably fluconazole (the most commonly used drug against candidiasis), in planktonic testing, which correlated with results in vivo. This raised the question whether activity shown by NZ alone against germ tubes would be reflected in drug combinations, and even whether synergy testing against germ tubes might be a better correlate of synergy in future in vivo studies. We show in this study significant NZ synergy with fluconazole against germ tubes, for several C. albicans isolates, with testing in many drug ratios. This observation opens the way for further explorations of this method of susceptibility testing for synergy, and correlation with combination therapy against candidiasis.

Genome-wide analysis of PTR transporters in Candida species and their functional characterization in Candida auris.

The peptide transport (PTR) or proton-dependent oligopeptide transporter (POT) family exploits the inwardly directed proton motive force to facilitate the cellular uptake of di/tripeptides. Interestingly, some representatives are also shown to import peptide-based antifungals in certain Candida species. Thus, the identification and characterization of PTR transporters serve as an essential first step for their potential usage as antifungal peptide uptake systems. Herein, we present a genome-wide inventory of the PTR transporters in five prominent Candida species. Our study identifies 2 PTR transporters each in C. albicans and C. dubliniensis, 1 in C. glabrata, 4 in C. parapsilosis, and 3 in C. auris. Notably, despite all representatives retaining the conserved features seen in the PTR family, there exist two distinct classes of PTR transporters that differ in terms of their sequence identities and lengths of certain extracellular and intracellular segments. Further, we also evaluated the contribution of each PTR protein of the newly emerged multi-drug-resistant C. auris in di/tripeptide uptake. Notably, deletion of two PTR genes BNJ08_003830 and BNJ08_005124 led to a marked reduction in the transport capabilities of several tested di/tripeptides. However, all three genes could complement the role of native PTR2 gene of Saccharomyces cerevisiae, albeit to varied levels. Besides, BNJ08_005124 deletion also resulted in increased resistance toward the peptide-nucleoside drug Nikkomycin Z as well as the glucosamine-6-phosphate synthase inhibitor, L-norvalyl-N3-(4-methoxyfumaroyl)-L-2,3-diaminopropionoic acid (Nva-FMDP), pointing toward its predominant role in their uptake mechanism. Altogether, the study provides an important template for future structure-function investigations of PTR transporters in Candida species. KEY POINTS: • Candida genome encodes for two distinct classes of PTR transporters. • Candida auris encodes for 3 PTR transporters with different specificities. • BNJ08_005124 in C. auris is involved in the uptake of Nikkomycin Z and Nva-FMDP.

Nikkomycin Z against Disseminated Coccidioidomycosis in a Murine Model of Sustained-Release Dosing.

Nikkomycin Z (nikZ) is a chitin synthase inhibitor. Efficacy against Coccidioides has been demonstrated in animal models of pulmonary or brain infection. Its short half-life in mice and in humans would necessitate divided daily dosing. We assayed nikZ efficacy in disseminated coccidioidomycosis (in a reduction of CFU design) and whether sustained release might be useful. Mice were challenged intravenously with low or high arthroconidial inocula. Fluconazole, clinically the most commonly used anticoccidioidal drug, was compared (gavage) at high dose to a dose range of nikZ administered intraperitoneally or, to mimic sustained release, administered continuously in drinking water. Therapy was given for 5 days. In vitro, both fluconazole and nikZ inhibited the isolate studied; nikZ was fungicidal. Oral nikZ therapy gave similar results to intraperitoneal nikZ and sterilized infection in most animals after low-inoculum challenge. In both challenges, oral nikZ produced greater reduction of CFU in organs (lung, liver, and spleen) than fluconazole. Oral nikZ doses of ≥200 mg/kg of body weight/day were particularly effective in all organs and were well tolerated. This efficacy occurred even though, after severe challenge, mice had reduced water intake, resulting in ingesting less than the desired dose, particularly initially after infection. This study shows, for the first time, efficacy of nikZ against disseminated coccidioidomycosis. Efficacy was shown after challenges producing different levels of severity of disease. This study also suggests the likely benefits of developing an extended release formulation supplying continuous systemic concentrations of nikZ.

Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species. A New Potential Target for Therapy.

N-acetylglucosamine (GlcNAc) serves as an essential structural sugar on the cell surface of organisms. For example, GlcNAc is a major component of bacterial peptidoglycan, it is an important building block of fungal cell walls, including a major constituent of chitin and mannoproteins, and it is also required for extracellular matrix generation by animal cells. Herein, we provide evidence for a uridine diphospho (UDP)-GlcNAc pathway in Pneumocystis species. Using an in silico search of the Pneumocystis jirovecii and P. murina (Pm) genomic databases, we determined the presence of at least four proteins implicated in the Saccharomyces cerevisiae UDP-GlcNAc biosynthetic pathway. These genes, termed GFA1, GNA1, AGM1, and UDP-GlcNAc pyrophosphorylase (UAP1), were either confirmed to be present in the Pneumocystis genomes by PCR, or, in the case of Pm uap1 (Pmuap1), functionally confirmed by direct enzymatic activity assay. Expression analysis using quantitative PCR of Pneumocystis pneumonia in mice demonstrated abundant expression of the Pm uap1 transcript. A GlcNAc-binding recombinant protein and a novel GlcNAc-binding immune detection method both verified the presence of GlcNAc in P. carinii (Pc) lysates. Studies of Pc cell wall fractions using high-performance gas chromatography/mass spectrometry documented the presence of GlcNAc glycosyl residues. Pc was shown to synthesize GlcNAc in vitro. The competitive UDP-GlcNAc substrate synthetic inhibitor, nikkomycin Z, suppressed incorporation of GlcNAc by Pc preparations. Finally, treatment of rats with Pneumocystis pneumonia using nikkomycin Z significantly reduced organism burdens. Taken together, these data support an important role for GlcNAc generation in the cell surface of Pneumocystis organisms.

Effects of Echinocandins in Combination with Nikkomycin Z against Invasive Candida albicans Bloodstream Isolates and the fks Mutants.

We evaluated the in vitro and in vivo effects of nikkomycin Z combined with an echinocandin (anidulafungin or micafungin) against two Candida albicans isolates and their lab-derived echinocandin-resistant fks mutants with FKS1 S645Y and FKS1 S645P. Synergistic effects were observed in all tested strains (fractional inhibitory concentration index, <0.5). Enhanced survival was observed in an immunocompromised murine model (log-rank test, P < 0.02). Our study demonstrated the therapeutic potential of nikkomycin Z-echinocandin combinations in managing echinocandin resistance.

Modeling nikkomycin Z dosing and pharmacology in murine pulmonary coccidioidomycosis preparatory to phase 2 clinical trials.

Nikkomycin Z (NikZ) is a chitin synthase inhibitor with activity against Coccidioides species that is being developed as a first-in-class orphan product for treatment of coccidioidomycosis. It has previously been shown to reduce lethal respiratory infections in mice to undetectable levels when treatment is begun 48 hours after infection. The studies described here focus on bracketing NikZ doses for phase 2 and 3 clinical trials, using an established mouse respiratory infection as a model and starting treatment 120 hours after infection. A dose of 80 mg/kg/day, divided into 2 doses, nearly eradicated infection, and larger doses did not improve fungal clearance. Increasing the duration of treatment from 1 week to 3 weeks resulted in a greater percentage of culture-negative mice. Comparative data show that plasma levels of NikZ that nearly eradicate Coccidioides in mice are achievable in patients and provide a plausibly effective dose range for initial phase 2 clinical studies.

Chitin synthase B: a midgut-specific gene induced by insect hormones and involved in food intake in Bombyx mori larvae.

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third-instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20-hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry-leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin-synthesis-dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae.

Polymycovirus Infection Sensitizes Aspergillus fumigatus for Antifungal Effects of Nikkomycin Z.

Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) weakens resistance of Aspergillus fumigatus common reference strain Af293 biofilms in intermicrobial competition with Pseudomonas aeruginosa. We compared the sensitivity of two infected and one virus-free Af293 strains to antifungal drugs. All three were comparably sensitive to drugs affecting fungal membranes (voriconazole, amphotericin) or cell wall glucan synthesis (micafungin, caspofungin). In contrast, forming biofilms of virus-free Af293 were much more resistant than AfuPmV-1-infected Af293 to nikkomycin Z (NikZ), a drug inhibiting chitin synthase. The IC50 for NikZ on biofilms was between 3.8 and 7.5 µg/mL for virus-free Af293 and 0.94-1.88 µg/mL for infected strains. The IC50 for the virus-free A. fumigatus strain 10AF was ~2 µg/mL in most experiments. NikZ also modestly affected the planktonic growth of infected Af293 more than the virus-free strain (MIC 50%, 2 and 4 µg/mL, respectively). Virus-free Af293 biofilm showed increased metabolism, and fungus growing as biofilm or planktonically showed increased growth compared to infected; these differences do not explain the resistance of the virus-free fungus to NikZ. In summary, AfuPmV-1 infection sensitized A. fumigatus to NikZ, but did not affect response to drugs commonly used against A. fumigatus infection. Virus infection had a greater effect on NikZ inhibition of biofilm than planktonic growth.

Chitin contributes to the formation of a feeding structure in a predatory nematode.

Some nematode predators and parasites form teeth-like denticles that are histologically different from vertebrate teeth, but their biochemical composition remains elusive. Here, we show a role of chitin in the formation of teeth-like denticles in Pristionchus pacificus, a model system for studying predation and feeding structure plasticity. Pristionchus forms two alternative mouth morphs with one tooth or two teeth, respectively. The P. pacificus genome encodes two chitin synthases, with the highly conserved chs-2 gene being composed of 60 exons forming at least four isoforms. Generating CRISPR-Cas9-based gene knockouts, we found that Ppa-chs-2 mutations that eliminate the chitin-synthase domain are lethal. However, mutations in the C terminus result in viable but teethless worms, with severe malformation of the mouth. Similarly, treatment with the chitin-synthase inhibitor Nikkomycin Z also results in teethless animals. Teethless worms can feed on various bacterial food sources but are incapable of predation. High-resolution transcriptomics revealed that Ppa-chs-2 expression is controlled by the sulfatase-encoding developmental switch Ppa-eud-1. This study indicates a key role of chitin in the formation of teeth-like denticles and the complex feeding apparatus in nematodes.

Cloning, functional characterization and screening of potential inhibitors for Chilo partellus chitin synthase A using in silico, in vitro and in vivo approaches.

Chitin synthase (CHS) is one of the crucial enzymes that play an essential role in chitin synthesis during the molting process, and it is considered to be the specific target to control insect pests. Currently, there are no potent inhibitors available in the market, which specifically target this enzyme. Pyrimidine nucleoside peptide, nikkomycin Z, binds to nucleotide-binding sites of fungal and insect CHS. But, their mode of action is still fragmentary due to the lack of a 3Dstructure of CHS. Chilo partellus is a severe pest insect of major food crops such as maize and sorghum, in an attempt to target integument expressed cuticular CpCHS. The CpChsA cDNA was cloned, and subsequently, their developmental and tissue-specific expression was studied. The 3D structure of the CHS catalytic domain was modeled, after which natural compounds were screened using a virtual screening workflow and resulted in the identification of five hit molecules. Molecular dynamics simulations were performed to investigate the dynamics and interactions of hits with CpCHS. The obtained results revealed that the compounds kasugamycin, rutin and robinin could act as potent inhibitors of CpCHS. All three molecules were observed to significantly reduce the chitin production as validated using in vitro and in vivo studies. Thus, this study aims to provide a set of novel inhibitor molecules against CpCHS for controlling the pest population. Communicated by Ramaswamy H. Sarma.

Initial Results of the International Efforts in Screening New Agents against Candida auris.

BACKGROUND: Candida auris is an emergent fungal pathogen and a global concern, mostly due to its resistance to many currently available antifungal drugs. OBJECTIVE: Thus, in response to this challenge, we evaluated the in vitro activity of potential new drugs, diphenyl diselenide (PhSe)2 and nikkomycin Z (nikZ), alone and in association with currently available antifungals (azoles, echinocandins, and polyenes) against Candida auris. METHODS: Clinical isolates of C. auris were tested in vitro. (PhSe)2 and nikZ activities were tested alone and in combination with amphotericin B, fluconazole, or the echinocandins, micafungin and caspofungin. RESULTS: (PhSe)2 alone was unable to inhibit C. auris, and antagonism or indifferent effects were observed in the combination of this compound with the antifungals tested. NikZ appeared not active alone either, but frequently acted cooperatively with conventional antifungals. CONCLUSION: Our data show that (PhSe)2 appears to not have a good potential to be a candidate in the development of new drugs to treat C. auris, but that nikZ is worthy of further study.

In silico Binding Profile Analysis and In vitro Investigation on Chitin Synthase Substrate and Inhibitors from Maize Stem Borer, Chilo partellus.

INTRODUCTION: Insect growth and metamorphosis are strictly dependent on the structural changes that occur in chitin containing tissues and organs. Chitin synthase catalyzes chitin polymerization by ß-(1, 4) glycosidic linkage of N-acetyl-D-glucosamine (GlcNAc) monomers; the major component of insect cuticles. Targeting this enzyme could be a promising strategy to control insect pests while avoiding adverse effects on coexisting populations. Nikkomycin Z and polyoxins are commercially available fungal inhibitors known to bind to the nucleotide-binding sites of insects and fungal chitin synthase. But the binding mode of chitin synthase has not been explored to date as its structure is not available yet. METHODS: To understand the structural features of the Chilo partellus chitin synthase enzyme (CpCHS), the three-dimensional (3D) structure of the CpCHS catalytic domain was modeled using ROBETTA webserver. The obtained model was used to investigate the binding mode of its substrate, uridine diphosphate-N-acetyl-D-glucosamine (UDP-GlcNAc), and inhibitors (nikkomycin Z and polyoxins) by molecular docking approach using Schrödinger Suite-Maestro v9.2. The docked complexes were further investigated for their interaction stability by performing molecular dynamics (MD) simulations using GROMACS v5.1.2. RESULTS: Our study highlighted the significance of various interactions made by CHS residues present in the Walker-B loop and donor-binding motifs with the substrate (UDP-GlcNAc), and GEDR motif with an acceptor (GlcNAc). Also, the interactions of the QRRRW motif while forming chitin polymer were explored. We observed that the inhibitors exhibited good binding affinity with these motifs, indicated by their docking and binding affinity scores. CONCLUSION: In vitro analysis suggested that nikkomycin Z showed higher inhibition of chitin synthase activity at a concentration of 2.5 µg.L-1. Our study provided insights into the crucial interactions of chitin synthase while designing inhibitors against insect pests.

Nikkomycin Z-Ready to Meet the Promise?

Nikkomycin Z (NikZ) has fungicidal activity against some fungal species which currently requires patients to endure chronic therapy, sometimes for years. This review highlights reports of NikZ activity against fungal species for which current therapeutics are still inadequate, as a potential roadmap for continuing investigation. The possibility of faster and more complete clinical resolution by using NikZ has attracted scientific attention for decades. NikZ inhibits chitin structure formation, which is important for fungi, but not found in mammals. NikZ raised no safety concerns in a human Phase 1 trial or in extensive toxicology studies. NikZ showed strong clinical benefit in dogs with natural Coccidioides infection. NikZ has protected animals against fatal infections of Candida albicans. NikZ provides high protection in synergistic combination with several agent classes against Candida and Aspergillus species.

Identification and Characterization of the Chitin Synthase Genes From the Fish Pathogen Saprolegnia parasitica.

Saprolegnia parasitica is a pathogenic oomycete responsible for severe fish infections. Despite its low abundance in the cell wall of S. parasitica, chitin is essential for hyphal growth as the inhibition of its biosynthesis leads to highly reduced growth. Here we identified and characterized chitin synthases (CHS) from S. parasitica as potential targets for anti-oomycete drugs. Bioinformatics analyses allowed the identification of six different putative Chs genes in the genome of the pathogen. The total number of genes was confirmed by Southern blot analysis and their expression levels were determined by quantitative PCR. Four of the six Chs genes were expressed in the mycelium, while the two others exhibited undetectable levels of expression. The mycelium was highly sensitive to the addition of nikkomycin Z (NZ) in the culture medium, which led to a decreased amount of chitin in the cell wall by up to 40% in the conditions tested, and to the formation of abnormal branching structures in the hyphae. The presence of NZ increased the expression level of one of the genes, Chs3, suggesting that the corresponding product is compensating the disruption of chitin biosynthesis in the hyphae. In addition, the activity of isolated CHS was strongly inhibited by NZ in vitro. Altogether our data indicate the importance of CHS for the vegetative growth of S. parasitica and demonstrate that these enzymes represent promising targets for the control of diseases caused by oomycetes.

Systemic Antifungal Agents: Current Status and Projected Future Developments.

By definition, an antifungal agent is a drug that selectively destroys fungal pathogens with minimal side effects to the host. Despite an increase in the prevalence of fungal infections particularly in immunocompromised patients, only a few classes of antifungal drugs are available for therapy, and they exhibit limited efficacy in the treatment of life-threatening infections. These drugs include polyenes, azoles, echinocandins, and nucleoside analogs. This chapter focuses on the currently available classes and representatives of systemic antifungal drugs in clinical use. We further discuss the unmet clinical needs in the antifungal research field; efforts in reformulation of available drugs such as Amphotericin B nanoparticles for oral drug delivery; development of new agents of known antifungal drug classes, such as albaconazole, SCY-078, and biafungin; and new drugs with novel targets for treatment of invasive fungal infections, including nikkomycin Z, sordarin derivatives, VT-1161 and VT-1129, F901318, VL-2397, and T-2307.

2,3-Butanedione monoxime increases sensitivity to Nikkomycin Z in the budding yeast Saccharomyces cerevisiae.

Nikkomycin Z (NZ) is a competitive inhibitor of chitin synthase III in the yeast Saccharomyces cerevisiae. Myosin type II-deficient yeast strains (myo1) display a dramatic reduction in growth when chitin synthase III activity is inhibited by NZ, supporting the contention that actomyosin motility plays an important role in maintaining cell wall integrity. A proposed inhibitor of cortical actin polymerization in vitro, 2,3-butanedione monoxime (BDM), also inhibits growth of wild-type yeast strains at a concentration of 20 mM. In this study, we assayed for potential in vivo interplay between BDM-sensitive cell functions and cell wall chitin synthesis by testing for increased sensitivity to NZ during co-treatment with BDM at sub-inhibitory concentrations. Our results show that BDM can increase the sensitivity of yeast cells to Nikkomycin Z.

Nikkomycin Z is an effective inhibitor of the chytrid fungus linked to global amphibian declines.

Fungal infections in humans, wildlife, and plants are a growing concern because of their devastating effects on human and ecosystem health. In recent years, populations of many amphibian species have declined, and some have become extinct due to chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis. For some endangered amphibian species, captive colonies are the best intermediate solution towards eventual reintroduction, and effective antifungal treatments are needed to cure chytridiomycosis and limit the spread of this pathogen in such survival assurance colonies. Currently, the best accepted treatment for infected amphibians is itraconazole, but its toxic side effects reduce its usefulness for many species. Safer antifungal treatments are needed for disease control. Here, we show that nikkomycin Z, a chitin synthase inhibitor, dramatically alters the cell wall stability of B. dendrobatidis cells and completely inhibits growth of B. dendrobatidis at 250 µM. Low doses of nikkomycin Z enhanced the effectiveness of natural antimicrobial skin peptide mixtures tested in vitro. These studies suggest that nikkomycin Z would be an effective treatment to significantly reduce the fungal burden in frogs infected by B. dendrobatidis.

A practical and scalable manufacturing process for an anti-fungal agent, Nikkomycin Z.

A scalable and reliable manufacturing process for Nikkomycin Z HCl on a 170 g scale has been developed and optimized. The process is characterized by a 2.3 g/L fermentation yield, 79% purification yield, and >98% relative purity of the final product. This method is suitable for further scale up and cGMP production. The Streptomyces tendae ΔNikQ strain developed during the course of this study is superior to any previously reported strain in terms of higher yield and purity of Nikkomycin Z.