Choi SR et al. (2019) ACS Infect Dis "Dual Inhibition of Klebsiella pneumoniae and Pseudomonas aeruginosa Iron Metabolism using Gallium Porphyrin and Gallium Nitrate."

Choi SR, Narayanasamy P, Britigan BE

[ACS Infect Dis, 2019]

Iron- and heme-uptake pathways and metabolism are promising targets for the development of new antimicrobial agents, as their disruption would lead to nutritional iron starvation and inhibition of bacterial growth. Salts of gallium III (Ga), an iron mimetic metal, disrupt iron-dependent biological processes by binding iron-utilizing proteins and compete with iron for uptake by bacterial siderophore-mediated iron uptake systems. Ga porphyrins, heme mimetic complexes, disrupt heme-utilizing hemeproteins. Because Ga(NO3)3 and Ga porphyrin disrupt different pathways of bacterial ion acquisition/utilization, we hypothesized that if used in combination they would result in enhanced antimicrobial activity. Antimicrobial activity of Ga porphyrins (Ga protoporphyrin (GaPP) or Ga mesoporphyrin (GaMP)) alone and in combination with Ga(NO3)3 were evaluated against Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus (MRSA) under iron-limited conditions. The Ga porphyrin/Ga(NO3)3 combination demonstrated substantial synergism against K. pneumoniae, P. aeruginosa, and MRSA. Time-kill assays revealed that the synergistic combination of GaPP/Ga(NO3)3 was bacteriostatic against K. pneumoniae and MRSA and bactericidal against P. aeruginosa. The GaPP/Ga(NO3)3 combination significantly disrupted K. pneumoniae and P. aeruginosa biofilms on plasma-coated surfaces and increased the survival of C. elegans infected with K. pneumoniae or P. aeruginosa. When assessing the antibacterial activity of the Ga(III)/antibiotic combinations, GaPP or Ga(NO3)3/colistin combinations also showed synergistic activity against K. pneumoniae and P. aeruginosa. Our results demonstrate that GaPP and Ga(NO3)3 have significant synergistic effects against several important human bacterial pathogens by dual inhibition of iron/heme metabolism.

Goytain A et al. (2005) Physiol Genomics "Functional characterization of human SLC41A1, a Mg2+ transporter with similarity to prokaryotic MgtE Mg2+ transporters."

Quamme GA, Goytain A

[Physiol Genomics, 2005]

We have begun to identify and characterize genes that are differentially expressed with low magnesium. One of these sequences conformed to the solute carrier SLC41A1. Real-time RT-PCR of RNA isolated from renal distal tubule epithelial [mouse distal convoluted tubule (MDCT)] cells cultured in low-magnesium media relative to normal media and in the kidney cortex of mice maintained on low-magnesium diets compared with those animals consuming normal diets confirmed that the SLC41A1 transcript is responsive to magnesium. Mouse SLC41A1 was cloned from MDCT cells, expressed in Xenopus laevis oocytes, and studied with two-electrode voltage-clamp studies. When expressed in oocytes, SLC41A1 mediates saturable Mg2+ uptake with a Michaelis constant of 0.67 mM. Transport of Mg2+ by SLC41A1 is rheogenic, voltage dependent, and not coupled to Na+ or Cl-. Expressed SLC41A1 transports a range of other divalent cations: Mg2+, Sr2+, Zn2+, Cu2+, Fe2+, Co2+, Ba2+, and Cd2+. The divalent cations Ca2+, Mn2+, and Ni2+ and the trivalent ion Gd3+ did not induce currents nor did they inhibit Mg2+ transport. The nonselective cation La3+ abolished Mg2+ uptake. The SLC41A1 transcript is present in many tissues, notably renal epithelial cells, and is upregulated in some tissues with magnesium deficiency. These studies suggest that SLC41A1 is a regulated Mg2+ transporter that might be involved in magnesium homeostasis in epithelial cells.

Devaney E et al. (2005) Int J Parasitol "Hsp90 is essential in the filarial nematode Brugia pahangi."

Devaney E, O'Neill K, Harnett W, Whitesell L, Kinnaird JH

[Int J Parasitol, 2005]

The development of a compound with activity against filarial nematodes (a ''macrofilaricide'') has been a long-standing goal of the World Health Organization. However, adult filariae have proved remarkably difficult to kill. To some extent this reflects a lack of understanding of key pathways and processes in filarial nematodes that may be suitable targets for chemotherapy. In this paper we show that geldanamycin (GA), a specific inhibitor of the activity of the heat shock protein 90 (Hsp90) family, kills adult worms and microfilariae (Mf) of Brugia pahangi at nanomolar concentrations. In addition, release of Mf from adult worms is inhibited within 24 h of exposure to GA and is not recoverable, demonstrating that GA effectively sterilises the worm. Similar results were obtained with a second filarial worm Acanthocheilonema viteae. In contrast GA has no effect on the free-living nematode Caenorhabditis elegans despite a high degree of conservation between the nematode Hsp90 sequences. In keeping with these findings, Brugia Hsp90 binds GA in a solid phase pull-down assay while the binding of C. elegans Hsp90 to immobilised GA is undetectable. In other eukaryotes, GA is known to bind in the N-terminal ATP pocket of Hsp90, disrupting its interactions with client proteins which are then targeted for degradation via the proteasome pathway. Thus, Hsp90 or some of its client proteins may provide novel targets for the chemotherapy of filarial infection.

Goytain A et al. (2005) Biochem Biophys Res Commun "Functional characterization of the mouse [corrected] solute carrier, SLC41A2."

Goytain A, Quamme GA

[Biochem Biophys Res Commun, 2005]

We have recently demonstrated that the human solute carrier, SLC41A1, is a Mg 2+ transporter that is regulated by extracellular magnesium. A BLAST search found a closely related protein encoded by SLC41A2 that may have related functional properties. In order to determine the function of SLC41A2, the corresponding cRNA was expressed in Xenopus laevis oocytes and Mg2+ currents were determined under voltage-clamp conditions. Further, real-time RT-PCR was performed to determine if SLC41A2 expression is regulated by magnesium. When expressed in oocytes, SLC41A2 mediates voltage-dependent and saturable Mg2+ uptake with a Michaelis constant of 0.34+/-0.05 mM. Expressed SLC41A2 transports a range of other divalent cations: Ba2+, Ni2+, Co2+, Fe2+, or Mn2+, but not Ca2+, Zn2+, or Cu2+. Mg2+ transport was inhibited by large concentrations of Ca2+. Real-time reverse transcription polymerase chain reaction of RNA isolated from renal distal tubule epithelial (MDCT) cells cultured in low-magnesium media relative to normal media and in kidney cortex of mice maintained on low-magnesium diets compared to those animals consuming normal diets showed that SLC41A2 transcript, unlike SLC41A1 mRNA, is not responsive to magnesium. These studies suggest that SLC41A2 is a Mg2+ transporter that might be involved in magnesium homeostasis in epithelial cells.

Li BW et al. (2011) PLoS Negl Trop Dis "Gender-associated genes in filarial nematodes are important for reproduction and potential intervention targets."

Rush AC, Li BW, Mitreva M, Jiang DJ, Weil GJ, Abubucker S

[PLoS Negl Trop Dis, 2011]

BACKGROUND: A better understanding of reproductive processes in parasitic nematodes may lead to development of new anthelmintics and control strategies for combating disabling and disfiguring neglected tropical diseases such as lymphatic filariasis and onchocerciasis. Transcriptomatic analysis has provided important new insights into mechanisms of reproduction and development in other invertebrates. We have performed the first genome-wide analysis of gender-associated (GA) gene expression in a filarial nematode to improve understanding of key reproductive processes in these parasites. METHODOLOGY/PRINCIPAL FINDINGS: The Version 2 Filarial Microarray with 18,104 elements representing 85% of the filarial genome was used to identify GA gene transcripts in adult Brugia malayi worms. Approximately 19% of 14,293 genes were identified as GA genes. Many GA genes have potential Caenorhabditis elegans homologues annotated as germline-, oogenesis-, spermatogenesis-, and early embryogenesis- enriched. The potential C. elegans homologues of the filarial GA genes have a higher frequency of severe RNAi phenotypes (such as lethal and sterility) than other C. elegans genes. Molecular functions and biological processes associated with GA genes were gender-segregated. Peptidase, ligase, transferase, regulator activity for kinase and transcription, and rRNA and lipid binding were associated with female GA genes. In contrast, catalytic activity from kinase, ATP, and carbohydrate binding were associated with male GA genes. Cell cycle, transcription, translation, and biological regulation were increased in females, whereas metabolic processes of phosphate and carbohydrate metabolism, energy generation, and cell communication were increased in males. Significantly enriched pathways in females were associated with cell growth and protein synthesis, whereas metabolic pathways such as pentose phosphate and energy production pathways were enriched in males. There were also striking gender differences in environmental information processing and cell communication pathways. Many proteins encoded by GA genes are secreted by Brugia malayi, and these encode immunomodulatory molecules such as antioxidants and host cytokine mimics. Expression of many GA genes has been recently reported to be suppressed by tetracycline, which blocks reproduction in female Brugia malayi. Our localization of GA transcripts in filarial reproductive organs supports the hypothesis that these genes encode proteins involved in reproduction. CONCLUSIONS/SIGNIFICANCE: Genome-wide expression profiling coupled with a robust bioinformatics analysis has greatly expanded our understanding of the molecular biology of reproduction in filarial nematodes. This study has highlighted key molecules and pathways associated with reproductive and other biological processes and identified numerous potential candidates for rational drug design to target reproductive processes.

David CL et al. (2003) Cell Stress Chaperones "Expression of a unique drug-resistant Hsp90 ortholog by the nematode Caenorhabditis elegans."

David CL, Whitesell L, Smith HE, Raynes DA, Pulcini EJ

[Cell Stress Chaperones, 2003]

In all species studied to date, the function of heat shock protein 90 (Hsp90), a ubiquitous and evolutionarily conserved molecular chaperone, is inhibited selectively by the natural product drugs geldanamycin (GA) and radicicol. Crystal structures of the N-terminal region of yeast and human Hsp90 have revealed that these compounds interact with the chaperone in a Bergerat-type adenine nucleotide-binding fold shared throughout the gyrase, Hsp90, histidine kinase mutL (GHKL) superfamily of adenosine triphosphatases. To better understand the consequences of disrupting Hsp90 function in a genetically tractable multicellular organism, we exposed the soil-dwelling nematode Caenorhabditis elegans to GA under a variety of conditions designed to optimize drug uptake. Mutations in the gene encoding C elegans Hsp90 affect larval viability, dauer development, fertility, and life span. However, exposure of worms to GA produced no discernable phenotypes, although the amino acid sequence of worm Hsp90 is 85% homologous to that of human Hsp90. Consistent with this observation, we found that solid phase-immobilized GA failed to bind worm Hsp90 from worm protein extracts or when translated in a rabbit reticulocyte lysate system. Further, affinity precipitation studies using chimeric worm-vertebrate fusion proteins or worm C-terminal truncations expressed in reticulocyte lysate revealed that the conserved nucleotide-binding fold of worm Hsp90 exhibits the novel ability to bind adenosine triphosphate but not GA. Despite its unusual GA resistance, worm Hsp90 appeared fully functional when expressed in a vertebrate background. It heterodimerized with its vertebrate counterpart and showed no-evidence of compromising its essential cellular functions. Heterologous expression of worm Hsp90 in tumor cells, however, did not render them GA resistant. These findings provide new insights into the nature of unusual N-terminal nucleotide-binding fold of Hsp90 and suggest that arget-related drug resistance is unlikely to emerge in patients receiving GA-like chemotherapeutic agents.

Tyagi R et al. (2019) Curr Top Med Chem "In Vitro and In Silico Studies of Glycyrrhetinic acid derivatives as Anti-Filarial Agents."

Srivastava SK, Mishra S, Tyagi R, Verma S, Khan F, Srivastava M, Alam S

[Curr Top Med Chem, 2019]

BACKGROUND: Lymphatic filariasis continues to be one of the major health problems in tropical and subtropical countries despite the use of standard drugs diethylcarbamazine and ivermectin because they are microfilaricides with poor or no activity on adult parasites. Therefore, new leads with activity on adult parasites are highly desirable. OBJECTIVE: Anti-filarial lead optimization by semi-synthetic modification of glycyrrhetinic acid (GA). METHODS: The GA was first converted into 3-O-acyl derivative, which was further converted into 12 amide derivatives. All these derivatives were assessed for their antifilarial potential by parasite motility assay. The binding affinity of active GA derivatives on trehalose-6-phosphate phosphatase (Bm-TPP) was assessed by molecular docking studies. RESULTS: Among 15 GA derivatives, GAD-2, GAD-3, and GAD-4 were found more potent than the GA and standard drug DEC. These derivatives reduced the motility of Brugia malayi adult worms by up to 74% while the GA and DEC reduced only up to 49%. Further, GA and most of its derivatives exhibited two times more reduction in MTT assay when compared to the standard drug DEC. These derivatives also showed 100% reduction of microfilariae and good interactions with Bm-TPP protein. CONCLUSION: The present study suggests that 3-O-acyl and linear chain amide derivatives of glycyrrhetinic acid may be potent leads against B. malayi microfilariae and adult worms. These results may be of great help in developing QSAR model for optimizing a new class of antifilarial lead from a very common, inexpensive, and non toxic natural product.

Jiang D et al. (2012) Int J Parasitol "Multiplex proteomics analysis of gender-associated proteins in Brugia malayi."

Weil G, Li B, Malone J, Jiang D, Townsend R

[Int J Parasitol, 2012]

Gender-associated (GA) genes are important for the development and reproduction of filarial nematodes. Identification and characterization of GA genes may provide insight into major pathways and processes involved in development and reproduction. The recent completion of the Brugia malayi genome has provided a good foundation for proteomics studies. Multiplex protein labelling and two-dimensional difference in-gel electrophoresis (2D-DIGE) combined with MALDI-TOF/TOF tandem MS were used to identify GA proteins. Thirty male and 32 female associated proteins were identified in this study. Many of these GA genes have homologues in Caenorhabditis elegans (83% male and 69% female), and most of the homologues have severe RNA interference (RNAi) phenotypes (72% male and 55% female) in C. elegans. Functional analysis showed that the male-associated genes are enriched for energy production, metabolic processes and cytoskeleton, while the female-associated genes are enriched for RNA modification and transcription. GA genes encode many excreted/secreted proteins. In situ localization studies showed that GA genes are mainly expressed in reproductive organs, and this is further evidence for their involvement in reproduction. Improved understanding of the basic biology of filarial nematodes may lead to improved tools for prevention and treatment of filarial infections. This study combined proteomics, in situ hybridization (ISH) and bioinformatics in a systems biology approach to improve understanding of gender differences and key proteins involved in reproduction in filarial worms. Advanced proteomics methods and bioinformatics led to the identification of 62 GA proteins for B. malayi. ISH revealed that most of those GA genes are expressed during embryogenesis or spermatogenesis. ISH results were consistent with RNAi data for C. elegans that linked the homologues of the B. malayi proteins to gamete production and embryogenesis.

Him NA et al. (2009) BMC Evol Biol "Hsp-90 and the biology of nematodes."

Devaney E, Gillan V, Him NA, Emes RD, Maitland K

[BMC Evol Biol, 2009]

BACKGROUND: Hsp-90 from the free-living nematode Caenorhabditis elegans is unique in that it fails to bind to the specific Hsp-90 inhibitor, geldanamycin (GA). Here we surveyed 24 different free-living or parasitic nematodes with the aim of determining whether C. elegans Hsp-90 was the exception or the norm amongst the nematodes. We combined these data with codon evolution models in an attempt to identify whether hsp-90 from GA-binding and non-binding species has evolved under different evolutionary constraints. RESULTS: We show that GA-binding is associated with life history: free-living nematodes and those parasitic species with free-living larval stages failed to bind GA. In contrast, obligate parasites and those worms in which the free-living stage in the environment is enclosed within a resistant egg, possess a GA-binding Hsp-90. We analysed Hsp-90 sequences from fifteen nematode species to determine whether nematode hsp-90s have undergone adaptive evolution that influences GA-binding. Our data provide evidence of rapid diversifying selection in the evolution of the hsp-90 gene along three separate lineages, and identified a number of residues showing significant evidence of adaptive evolution. However, we were unable to prove that the selection observed is correlated with the ability to bind geldanamycin or not. CONCLUSION: Hsp-90 is a multi-functional protein and the rapid evolution of the hsp-90 gene presumably correlates with other key cellular functions. Factors other than primary amino acid sequence may influence the ability of Hsp-90 to bind to geldanamycin.

Sonobe Y et al. (2021) Nat Commun "A C. elegans model of C9orf72-associated ALS/FTD uncovers a conserved role for eIF2D in RAN translation."

Roos RP, Brown AEX, Aburas J, Fleming AC, Islam P, Gendron TF, Gao FB, Krishnan G, Kratsios P, Kankel MW, Kiskinis E, Sonobe Y, Gu Y, Warren EC, Ghadge G

[Nat Commun, 2021]

A hexanucleotide repeat expansion GGGGCC in the non-coding region of C9orf72 is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Toxic dipeptide repeats (DPRs) are synthesized from GGGGCC via repeat-associated non-AUG (RAN) translation. Here, we develop C. elegans models that express, either ubiquitously or exclusively in neurons, 75 GGGGCC repeats flanked by intronic C9orf72 sequence. The worms generate DPRs (poly-glycine-alanine [poly-GA], poly-glycine-proline [poly-GP]) and poly-glycine-arginine [poly-GR]), display neurodegeneration, and exhibit locomotor and lifespan defects. Mutation of a non-canonical translation-initiating codon (CUG) upstream of the repeats selectively reduces poly-GA steady-state levels and ameliorates disease, suggesting poly-GA is pathogenic. Importantly, loss-of-function mutations in the eukaryotic translation initiation factor 2D (eif-2D/eIF2D) reduce poly-GA and poly-GP levels, and increase lifespan in both C. elegans models. Our in vitro studies in mammalian cells yield similar results. Here, we show a conserved role for eif-2D/eIF2D in DPR expression.